Compositions and methods for treating collagen-mediated diseases

ABSTRACT

A drug product comprising a combination of highly purified collagenase I and collagenase II from  Clostridium histolyticum  is disclosed. The drug product includes collagenase I and collagenase II in a ratio of about 1 to 1, with a purity of greater than at least 95%. The invention further disclosed improved fermentation and purification processes for preparing the said drug product.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/613,882, filed Feb. 4, 2015 which is a continuation of U.S.application Ser. No. 13/713,019, filed Dec. 13, 2012, now abandoned,which is a continuation of U.S. application Ser. No. 13/463,142, filedon May 3, 2012, now abandoned, which is a continuation of U.S.application Ser. No. 13/084,722, filed on Apr. 12, 2011, now abandoned;which is a continuation of U.S. application Ser. No. 12/871,159, filedon Aug. 30, 2010, now abandoned; which is a continuation of U.S.application Ser. No. 11/699,302, filed on Jan. 29, 2007, now U.S. Pat.No. 7,811,560, issued on Oct. 12, 2010, which claims the benefit of U.S.Provisional Application No. 60/763,470 filed on Jan. 30, 2006 and U.S.Provisional Application No. 60/784,135, filed Mar. 20, 2006. The entireteachings of the above applications are incorporated herein byreference.

PARTIES TO A JOINT RESEARCH AGREEMENT

Auxilium Pharmaceuticals Inc. and BioSpecifics Technologies Corp. areparties to a “joint research agreement” as defined in 35 USC 103(c)(3).

BACKGROUND OF THE INVENTION

Collagen is the major structural constituent of mammalian organisms andmakes up a large portion of the total protein content of skin and otherparts of the animal body. In humans, it is particularly important in thewound healing process and in the process of natural aging. Various skintraumas such as burns, surgery, infection and accident are oftencharacterized by the erratic accumulation of fibrous tissue rich incollagen and having increased proteoglycan content. In addition to thereplacement of the normal tissue which has been damaged or destroyed,excessive and disfiguring deposits of new tissue sometimes form duringthe healing process. The excess collagen deposition has been attributedto a disturbance in the balance between collagen synthesis and collagendegradation.

Numerous diseases and conditions are associated with excess collagendeposition and the erratic accumulation of fibrous tissue rich incollagen. Such diseases and conditions are collectively referred toherein as “collagen-mediated diseases”. Collagenase has been used totreat a variety of collagen-mediated diseases. Collagenase is an enzymethat has the specific ability to digest collagen.

Collagenase for use in therapy may be obtained from a variety of sourcesincluding mammalian (e.g. human), crustacean (e.g. crab, shrimp),fungal, and bacterial (e.g. from the fermentation of Clostridium,Streptomyces, Pseudomonas, or Vibrio). Collagenase has also beengenetically engineered. One common source of crude collagenase is from abacterial fermentation process, specifically the fermentation of C.histolyticum (C. his). The crude collagenase obtained from C. his may bepurified using any of a number of chromatographic techniques.

One drawback of the fermentation process from C. his is that it yieldsuncertain ratios of the various collagenases such as collagenase I andcollagenase II, often used in therapeutic compositions to treat collagenmediated conditions. Further, the culture has historically required theuse of meat products. This meat culture was originally derived from theH4 strain of Clostridium histolyticum, Dr. I. Mandl's laboratory inColumbia University in 1956 and deposited with the ATCC. Lyophilizedvials were made out of the cooked meat culture and named as ABCClostridium histolyticum master cell bank.

Various ratios of collagenase I to collagenase II in a therapeuticcollagenase preparation have different biological effects. Therefore, atherapeutic collagenase preparation in which the ratio of collagenase Ito collagenase II in the preparation can be easily and efficientlydetermined and controlled to obtain superior, and consistent enzymeactivity and therapeutic effect, would be desirable.

SUMMARY OF THE INVENTION

The present invention provides a collagenase composition comprising acombination of highly purified collagenase I and collagenase II.Preferably, the collagenase I and collagenase II are present in a massratio of about 1 to 1. When used as a pharmaceutical composition fortreating collagen-mediated diseases, the composition of the inventionprovides improved and consistent therapeutic effect while lowering thepotential for side effects.

The invention further provides methods for preparing a collagenasecomposition of the invention, pharmaceutical formulations comprising acomposition of the invention and methods for treating patients sufferingfrom a collagen-mediated disease using a collagenase composition of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

FIG. 1 depicts growth curves (OD vs time) of C. histolyticum in 5 LDCFT24a,b fermentations.

FIG. 2 depicts net growth curves (Net OD vs time) of C. histolyticum in5 L DCFT24a,b fermentations.

FIG. 3 is a 8% Tris-glycine SDS PAGE gel from the second fermentation:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Collagenase I—0.27 μg    -   Lane 3: Collagenase II—0.29 μg    -   Lane 4: 20 h (6.12 μL of sample)—Harvest point    -   Lane 5: 19 h (6.12 μL of sample)    -   Lane 6: 17 h (6.12 μL of sample)    -   Lane 7: 16 h (6.12 μL of sample)    -   Lane 8: 15 h (6.12 μL of sample)    -   Lane 9: 14 h (6.12 μL of sample)    -   Lane 10: 13 h (6.12 μL of sample)    -   Lane 11: 11.6 h-19 h (6.12 μL of sample)    -   Lane 12: 10.5 h (6.12 μL of sample).

FIG. 4 is a 8% Tris-glycine SDS PAGE gel from the first fermentation:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Collagenase I—0.27 μg    -   Lane 3: Collagenase II—0.29 μg    -   Lane 4: 20 h (6.12 μL of sample)—Harvest point    -   Lane 5: 19 h (6.12 μL of sample)    -   Lane 6: 17 h (6.12 μL of sample)    -   Lane 7: 16 h (6.12 μL of sample)    -   Lane 8: 15 h (6.12 μL of sample)    -   Lane 9: 14 h (6.12 μL of sample)    -   Lane 10: 13 h (6.12 μL of sample)    -   Lane 11: 11.4 h (6.12 μL of sample)    -   Lane 12: 10.4 h (6.12 μL of sample).

FIG. 5 is a Semi-quantitative SDS PAGE gel for the second fermentation,harvest point sample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.87 μL of sample (1/7 dilution of fermentation sample)    -   Lane 3: 1.22 μL of sample (1/5 dilution of fermentation sample)    -   Lane 4: 1.53 μL of sample (1/4 dilution of fermentation sample)    -   Lane 5: 2.04 μL of sample (1/3 dilution of fermentation sample)    -   Lane 6: 0.27 μg collagenase I    -   Lane 7: 0.18 μg collagenase I    -   Lane 8: 0.135 μg collagenase I    -   Lane 9: 0.29 μg collagenase II    -   Lane 10: 0.193 μg collagenase II    -   Lane 11: 0.145 μg collagenase II.

FIG. 6 represents fermentation strategy used for DCFT26a and DCFT26b.

FIG. 7 depicts growth curves (OD vs time) of C. histolyticum in 5 LDCFT26a,b fermentations.

FIG. 8 depicts net growth curves (Net OD vs time) of C. histolyticum in5 L DCFT26a,b fermentations.

FIG. 9 is a SDS PAGE gel for DCFT26a:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Collagenase I—0.67 μg    -   Lane 3: Collagenase II—0.72 μg    -   Lane 4: 20 h (6.12 μL of sample)—Harvest Point    -   Lane 5: 19 h (6.12 μL of sample)    -   Lane 6: 18 h (6.12 μL of sample)    -   Lane 7: 17 h (6.12 μL of sample)    -   Lane 8: 16 h (6.12 μL of sample)    -   Lane 9: 14 h (6.12 μL of sample)    -   Lane 10: 13 h (6.12 μL of sample)    -   Lane 11: 11 h (6.12 μL of sample).

FIG. 10 is a SDS PAGE gel for DCFT26b:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 20 h (6.12 μL of sample)—Harvest point    -   Lane 3: 19 h (6.12 μL of sample)    -   Lane 4: 18 h (6.12 μL of sample)    -   Lane 5: 17 h (6.12 μL of sample)    -   Lane 6: 16 h (6.12 μL of sample)    -   Lane 7: 15 h (6.12 μL of sample)    -   Lane 8: 14 h (6.12 μL of sample)    -   Lane 9: 13 h (6.12 μL of sample)    -   Lane 10: 11 h (6.12 μL of sample)    -   Lane 11: Collagenase I—0.67 μg    -   Lane 12: Collagenase II—0.72 μg.

FIG. 11 is a semi-quantitative SDS PAGE gel for DCFT26a, harvest pointsample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I    -   Lane 3: 0.18 μm collagenase I    -   Lane 4: 0.135 μm collagenase I    -   Lane 5: 0.29 μg collagenase II    -   Lane 6: 0.193 μm collagenase II    -   Lane 7: 0.145 μm collagenase II    -   Lane 8: 0.87 μL of sample (1/7 dilution of fermentation sample)    -   Lane 9: 1.22 μL of sample (1/5 dilution of fermentation sample)    -   Lane 10: 1.53 μL of sample (1/4 dilution of fermentation sample)    -   Lane 11: 2.04 μL of sample (1/3 dilution of fermentation        sample).

FIG. 12 is a Semi-quantitative SDS PAGE gel for DCFT26b, harvest pointsample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I    -   Lane 3: 0.18 μm collagenase I    -   Lane 4: 0.135 μm collagenase I    -   Lane 5: 0.29 μg collagenase II    -   Lane 6: 0.193 μm collagenase II    -   Lane 7: 0.145 μm collagenase II    -   Lane 8: 2.04 μL of sample (1/3 dilution of fermentation sample)    -   Lane 9: 1.53 μL of sample (1/4 dilution of fermentation sample)    -   Lane 10: 1.22 μL of sample (1/5 dilution of fermentation sample)    -   Lane 11: 0.87 μL of sample (1/7 dilution of fermentation        sample).

FIG. 13 is a SDS PAGE gel for post-dialysed ammonium sulphateprecipitated (100 g/L and 150 g/L) samples, DCFT26a, harvest pointsample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.67 μg collagenase I and 0.72 μg collagenase II    -   Lane 3: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 4: 6.12 μL of supernatant sample from SC11    -   Lane 5: post dialysed sample—100 g/L AS (Neat)    -   Lane 6: post dialysed sample—100 g/L AS (1/5)    -   Lane 7: post dialysed sample—100 g/L AS (1/10)    -   Lane 8: post dialysed sample—150 g/L AS (Neat)    -   Lane 9: post dialysed sample—150 g/L AS (1/5)    -   Lane 10: post dialysed sample—150 g/L AS (1/10).

FIG. 14 is a SDS PAGE gel for post-dialysed ammonium sulphateprecipitated (200 g/L and 250 g/L) samples, DCFT26a, harvest point:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.67 μg collagenase I and 0.72 μg collagenase II    -   Lane 3: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 4: 6.12 μL of supernatant sample from SC11    -   Lane 5: post dialysed sample—200 g/L AS (Neat)    -   Lane 6: post dialysed sample—200 g/L AS (1/5)    -   Lane 7: post dialysed sample—200 g/L AS (1/10)    -   Lane 8: post dialysed sample—250 g/L AS (Neat)    -   Lane 9: post dialysed sample—250 g/L AS (1/5)    -   Lane 10: post dialysed sample—250 g/L AS (1/10).

FIG. 15 is a SDS PAGE gel for post-dialysed ammonium sulphateprecipitated (300 g/L and 400 g/L) samples, DCFT26a, harvest point:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.67 μg collagenase I and 0.72 μg collagenase II    -   Lane 3: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 4: 6.12 μL of supernatant sample from SC11    -   Lane 5: post dialysed sample—300 g/L AS (Neat sample)    -   Lane 6: post dialysed sample—300 g/L AS (1/5 dilution)    -   Lane 7: post dialysed sample—300 g/L AS (1/10 dilution)    -   Lane 8: post dialysed sample—400 g/L AS (Neat)    -   Lane 9: post dialysed sample—4000 g/L AS (1/5 dilution)    -   Lane 10: post dialysed sample—400 g/L AS (1/10 dilution).

FIG. 16 depicts a Growth curves (OD vs time and net OD vs time) of C.histolyticum in PBFT57 fermentation.

FIG. 17 is a Semi-quantitative SDS PAGE gel, harvest point sample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I    -   Lane 3: 0.18 μm collagenase I    -   Lane 4: 0.135 μg collagenase I    -   Lane 5: 0.29 μg collagenase II    -   Lane 6: 0.193 μm collagenase II    -   Lane 7: 0.145 μm collagenase II    -   Lane 8: 2.04 μL of sample (1/3 dilution of fermentation harvest        sample)    -   Lane 9: 1.53 μL of sample (1/4 dilution of fermentation harvest        sample)    -   Lane 10: 1.22 μL of sample (1/5 dilution of fermentation harvest        sample)    -   Lane 11: 0.87 μL of sample (1/7 dilution of fermentation harvest        sample).

FIG. 18a is a quantitative SDS PAGE gel for post-dialysed 500 mL samplefrom fermentation PBFT57, harvest point sample. 400 g/L of ammoniumsulphate added:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.272 μg collagenase I and 0.286 μg collagenase II    -   Lane 3: 0.181 μg collagenase I and 0.190 μg collagenase II    -   Lane 4: 0.136 μm collagenase I and 0.142 μg collagenase II    -   Lane 5: 0.109 μm collagenase I and 0.114 μg collagenase II    -   Lane 6: post dialysed sample—400 g/L AS (1/15 dilution)    -   Lane 7: post dialysed sample—400 g/L AS (1/20 dilution)    -   Lane 8: post dialysed sample—400 g/L AS (1/25 dilution)    -   Lane 9: post dialysed sample—400 g/L AS (1/30 dilution)    -   Lane 10: post dialysed sample—400 g/L AS (1/35 dilution)    -   Lane 11: High Molecular Weight Marker.

FIG. 18b is a SDS PAGE of the supernatants after centrifugation of theammonium sulphate precipitated samples:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μm Col I and 0.29 μg Col II    -   Lane 3: Supernatant (neat) of post ammonium sulphate        precipitated sample (400 g/L slow addition)    -   Lane 4: Supernatant (neat) of post ammonium sulphate        precipitated sample (400 g/L fast addition)    -   Lane 5: Supernatant (neat) of post ammonium sulphate        precipitated sample (440 g/L slow addition)    -   Lane 6: Supernatant (neat) of post ammonium sulphate        precipitated sample (480 g/L slow addition)    -   Lane 7: Supernatant (neat) of post ammonium sulphate        precipitated sample (520 g/L slow addition)    -   Lane 8: Supernatant (neat) of post ammonium sulphate        precipitated sample (400 g/L, pH 6)    -   Lane 9: Supernatant (neat) of post ammonium sulphate        precipitated sample (400 g/L, oxygenated).

FIG. 19 is a Semi-quantitative SDS PAGE gel showing diluted samples fromthe harvest point supernatant and the post dialysed ammonium sulphate(with 400 g/L—fast addition) precipitated sample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Fermentation sample—harvest (neat)    -   Lane 3: Fermentation sample—harvest (1/1 dilution)    -   Lane 4: Fermentation sample—harvest (1/2 dilution)    -   Lane 5: Fermentation sample—harvest (1/3 dilution)    -   Lane 6: Fermentation sample—harvest (1/4 dilution)    -   Lane 7: Post dialysed sample—harvest (1/17.54 dilution)        corresponds to lane 1    -   Lane 8: Post dialysed sample—harvest (1/35.08 dilution)        corresponds to lane 2    -   Lane 9: Post dialysed sample—harvest (1/52.62 dilution)        corresponds to lane 3    -   Lane 10: Post dialysed sample—harvest (1/70.16 dilution)        corresponds to lane 4    -   Lane 11: Post dialysed sample—harvest (1/87.70 dilution)        corresponds to lane 5.

FIG. 20 is a semi-quantitative SDS PAGE gel for PBFT57 showing dilutedsamples from the harvest point supernatant and the post dialysedammonium sulphate (with 520 g/L) precipitated sample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Fermentation sample—harvest (neat)    -   Lane 3: Fermentation sample—harvest (1/1 dilution)    -   Lane 4: Fermentation sample—harvest (1/2 dilution)    -   Lane 5: Fermentation sample—harvest (1/3 dilution)    -   Lane 6: Fermentation sample—harvest (1/4 dilution)    -   Lane 7: Post dialysed sample—harvest (1/15.63) corresponds to        lane 1    -   Lane 8: Post dialysed sample—harvest (1/31.26) corresponds to        lane 2    -   Lane 9: Post dialysed sample—harvest (1/46.89) corresponds to        lane 3    -   Lane 10: Post dialysed sample—harvest (1/62.52) corresponds to        lane 4    -   Lane 11: Post dialysed sample—harvest (1/78.15) corresponds to        lane 5.

FIG. 21 depicts growth curves (Net OD vs time) of C. histolyticumstrains 004 and 013 in PBFT58c,d fermentations.

FIG. 22 is a SDS PAGE gel for PBFT58c (Strain 004):

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Collagenase I—1.00 μg    -   Lane 3: Collagenase I—0.67 μg    -   Lane 4: Collagenase II—1.08 μg    -   Lane 5: Collagenase II—0.72 μg    -   Lane 6: 16.25 h (6.12 μL of sample)    -   Lane 7: 17 h (6.12 μL of sample)    -   Lane 8: 18 h (6.12 μL of sample)    -   Lane 9: 19 h (6.12 μL of sample)    -   Lane 10: 20.5 h (6.12 μL of sample).

FIG. 23 is a SDS PAGE gel for PBFT58d (Strain 013):

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: Collagenase I—1.00 μg    -   Lane 3: Collagenase I—0.67 μg    -   Lane 4: Collagenase II—1.08 μg    -   Lane 5: Collagenase II—0.72 μg    -   Lane 6: 16.25 h (6.12 μL of sample)    -   Lane 7: 17 h (6.12 μL of sample)    -   Lane 8: 18 h (6.12 μL of sample)    -   Lane 9: 19 h (6.12 μL of sample)    -   Lane 10: 20.5 h (6.12 μL of sample).

FIG. 24 is a semi-quantitative SDS PAGE gel for PBFT58c (strain 004),harvest point sample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 3: 0.18 μg collagenase I and 0.19 μg collagenase II    -   Lane 4: 0.135 μg collagenase I and 0.145 μg collagenase II    -   Lane 5: 0.108 μg collagenase I and 0.116 μg collagenase II    -   Lane 6: 6.12 μL of sample    -   Lane 7: 3.06 μL of sample    -   Lane 8: 2.04 μL of sample    -   Lane 9: 1.53 μL of sample    -   Lane 10: 1.22 μL of sample.

FIG. 25 is a semi-quantitative SDS PAGE gel for PBFT58d (strain 013),harvest point sample:

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 3: 0.18 μg collagenase I and 0.19 μg collagenase II    -   Lane 4: 0.135 μg collagenase I and 0.145 μg collagenase II    -   Lane 5: 0.108 μm collagenase I and 0.116 μg collagenase II    -   Lane 6: 6.12 μL of sample    -   Lane 7: 3.06 μL of sample    -   Lane 8: 2.04 μL of sample    -   Lane 9: 1.53 μL of sample    -   Lane 10: 1.22 μL of sample.

FIG. 26 is SDS PAGE gel for post-dialysed harvest point sample (520 g/Lammonium sulphate) of PBFT58c fermentation (strain 004):

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 3: 0.18 μm collagenase I and 0.19 μg collagenase II    -   Lane 4: 0.135 μm collagenase I and 0.145 μg collagenase II    -   Lane 5: 0.108 μm collagenase I and 0.116 μg collagenase II    -   Lane 6: post dialysed harvest point sample—Neat    -   Lane 7: post dialysed harvest point sample—(1/5 dilution)    -   Lane 8: post dialysed harvest point sample—(1/10 dilution)    -   Lane 9: post dialysed harvest point sample—(1/15 dilution)    -   Lane 10: post dialysed harvest point sample—(1/20 dilution).

FIG. 27 is a SDS PAGE gel for post-dialysed harvest point sample (400g/L ammonium sulphate) of PBFT58d fermentation (strain 013):

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 0.27 μg collagenase I and 0.29 μg collagenase II    -   Lane 3: 0.18 μm collagenase I and 0.19 μg collagenase II    -   Lane 4: 0.135 μm collagenase I and 0.145 μg collagenase II    -   Lane 5: 0.108 μm collagenase I and 0.116 μg collagenase II    -   Lane 6: post dialysed harvest point sample—Neat    -   Lane 7: post dialysed harvest point sample—(1/5 dilution)    -   Lane 8: post dialysed harvest point sample—(1/10 dilution)    -   Lane 9: post dialysed harvest point sample—(1/15 dilution)    -   Lane 10: post dialysed harvest point sample—(1/20 dilution).

FIG. 28 is illustrates a flow chart of the Experimental procedure usedfor screening the alternative vegetable peptones.

FIG. 29 illustrates a fed-batch strategy for DCFT27a,b fermentations.

FIG. 30 depicts growth curves (Net OD vs time) of C. histolyticum in 5 LDCFT27a and DCFT27b fed-batch fermentations.

FIG. 31 depicts growth curves (Net OD vs time) of C. histolyticum in 5 LPBFT59a,b,c batch fermentations.

FIG. 32 depicts a growth curve (Net OD vs time) of C. histolyticum in 5L DCFT27d fed-batch fermentation.

FIG. 33a is a SDS PAGE gel for DCFT27d (Phytone supplemented with aminoacids):

-   -   Lane 1: High Molecular Weight Marker    -   Lane 2: 18 h (6.12 μL of sample)    -   Lane 3: 17 h (6.12 μL of sample)    -   Lane 4: 15 h (6.12 μL of sample)    -   Lane 5: 14 h (6.12 μL of sample)    -   Lane 6: 13 h (6.12 μL of sample)    -   Lane 7: 11.3 h (6.12 μL of sample)    -   Lane 8: 0.27 μg Collagenase I and 0.29 μg Collagenase II.

FIG. 33b represents a schematic diagram of the inoculation procedure.

FIG. 33c represents a flow chart of an approximately 200 L fed batchinoculation process.

FIG. 34 shows a chromatogram after hydroxyapatite chromatography.

FIG. 35 shows a chromatogram after a fractogel TMAE anion exchange.

FIG. 36 is an 8% Tris-Glycine SDS-PAGE analysis of Pre HA, Post HA andPost TMAE material from 5 L scale process:

Lane Sample Load volume, μl 1 High Molecular Weight Marker 20 2Collagenase ABC I reference 1 μg 20 3 Collagenase ABC II reference 1 μg20 4 Pre HA - 1 μg 20 5 Post HA - 1 μg 20 6 Post TMAE - 1 μg 20.

FIG. 37 shows a chromatogram after a fractogel TMAE anion exchange.

FIG. 38 is an 8% Tris-Glycine SDS-PAGE analysis of Q Sepharose IEXchromatography of post TMAE material run in the presence leupeptin:

Lane Sample Load volume, μl 1 High Molecular Weight marker 20 2Collagenase ABC I reference 1 μg 20 3 Collagenase ABC II reference 1 μg20 4 Post TMAE/Post Dialysis - 1 μg 20 5 Fraction D6 - neat 20 6Fraction E6 - neat 20 7 Fraction F7 - 1 μg 20 8 Fraction F6 - 1 μg 20 9Fraction F5 - 1 μg 20 10 Fraction F4 - neat 20.

FIG. 39 is an 8% Tris-Glycine SDS-PAGE analysis of Q Sepharose IEXchromatography of post TMAE material run in the presence of leupeptin.Gel 2-Peak 2 (ABCI):

Lane SampleLoad volume, μl Load volume, μl 1 High Molecular Weightmarker 20 2 Collagenase ABC I reference 1 μg 20 3 Collagenase ABC IIreference 1 μg 20 4 Fraction B2 - neat 20 5 Fraction C1 - 1 μg 20 6Fraction C2 - 1 μg 20 7 Fraction C3 - 1 μg 20 8 Fraction C4 - 1 μg 20 9Fraction C5 - neat 20 10 Fraction C6 - neat 20.

FIG. 40 shows a chromatogram after a Q Sepharose HP anion exchange withmodified gradient.

FIG. 41 shows a chromatogram after a Superdex 75 Gel Permeationchromatography of ABCII.

FIG. 42 is a 12% Bis-Tris SDS-PAGE analysis of Superdex 75 GPC ofconcentrated ABC II run in the presence of arginine:

Lane Sample Load volume, μl 1 Mark 12 marker 10 2 Collagenase ABC Ireference 1 μg 15 3 Collagenase ABC II reference 1 μg 15 4 GPC load 1 μg15 5 Fraction D4 15 6 Fraction D3 15 7 Fraction D2 15 8 Fraction D1 Neat15 9 Fraction E1 15 10 Fraction E2 15 11 Fraction E3 15 12 Fraction E415.

FIG. 43 shows a chromatogram after a Superdex 75 Gel Permeationchromatography of ABCI.

FIG. 44 is a 4-12% Bis-Tris SDS-PAGE analysis of Superdex 75 GPC ofconcentrated ABC I run in the presence of arginine:

Lane Sample Load volume, μl 1 Mark 12 marker 10 2 Collagenase ABC Ireference 1 μg 15 3 Collagenase ABC II reference 1 μg 15 4 GPC load 1 μg15 5 Fraction D4 15 6 Fraction D3 15 7 Fraction D2 15 8 Fraction D1 15 9Fraction E1 15 10 Fraction E2 15 11 Fraction E3 15 12 Fraction E4 15.

FIG. 45 represents a flow chart of one proposed manufacturing process.

FIG. 46 represents a flow chart of the fermentation procedure forprocess 3.

FIG. 47 represents a flow chart of the purification procedure forprocess 3.

FIG. 48 is a SDS-PAGE (reduced) Coomasie stained for Intermediates AUXIand AUXII:

-   -   Lane    -   1. High Molecular Weight Markers    -   2. 0.132 mg/ml ABC-I Reference    -   3. 0.0265 mg/ml ABC-I Reference    -   4. 0.132 mg/ml AUX-I Intermediate    -   5. 0.0265 mg/ml AUX-I Intermediate    -   6. 0.132 mg/ml ABC-II Reference    -   7. 0.0265 mg/ml ABC-II Reference    -   8. 0.132 mg/ml AUX-II Intermediate    -   9. 0.0265 mg/ml AUX-II Intermediate.

FIG. 49 is a SDS-PAGE (reduced) Coomasie stained for Drug Substance:

-   -   Lane    -   1. High Molecular Weight Markers    -   2. 0.132 mg/ml Mixed BTC Reference    -   3. 0.0265 mg/ml Mixed BTC Reference    -   4. 0.132 mg/ml Drug Substance    -   5. 0.0265 mg/ml Drug Substance.

FIG. 50 is SDS-PAGE (reduced) Silver stained Drug Substance:

-   -   Lane    -   1. HMW marker    -   2. Mixed BTC reference 1.3 μg    -   3. Blank    -   4. Drug Substance 1.3 μg    -   5. Drug Substance 0.27 μg    -   6. Drug Substance 0.13 μg.

FIG. 51 depicts a comparison of C. histolyticum grown on ProteosePeptone #3 in a batch fermentation to the existing fermentation processusing Phytone peptone during fed-batch cultivation;

GCFT03a PP3 batch

GCFT03d Phytone fed-batch

FIG. 52 is a SDS-PAGE analysis of the collagenase product at the harvestpoint (20 h) of a 5 L Proteose Peptone #3 batch fermentation (GCFT03b)(8% Tris-Glycine):

Lane Sample 1 High Molecular Weight Marker 2  0.27 μg AUXI 3  0.18 μgAUXI 4 0.135 μg AUXI  5  0.29 μg AUXII 6 0.193 μg AUXII 7 0.145 μg AUXII8 0.87 μL of sample ( 1/7 dilution of fermentation sample) 9 1.22 μL ofsample (⅕ dilution of fermentation sample) 10 1.53 μL of sample (¼dilution of fermentation sample) 11 2.04 μL of sample (⅓ dilution offermentation sample) Estimates AUXI ~176 mg/L AUXII ~190 mg/L.

FIG. 53 is a SDS-PAGE analysis of the collagenase product at the harvestpoint (20 h) of a 5 L Phytone fed-batch fermentation (GCFT03d) (8%Tris-Glycine):

Lane Sample 1 High Molecular Weight Marker 2  0.27 μg AUXI 3  0.18 μgAUXI 4 0.135 μg AUXI  5  0.29 μg AUXII 6 0.193 μg AUXII 7 0.145 μg AUXII8 0.87 μL of sample ( 1/7 dilution of fermentation sample) 9 1.22 μL ofsample (⅕ dilution of fermentation sample) 10 1.53 μL of sample (¼dilution of fermentation sample) 11 2.04 μL of sample (⅓ dilution offermentation sample) Estimates AUXI ~88 mg/L AUXII ~142 mg/L.

FIG. 54 illustrates three fermentations of Clostridium histolyticumgrown on 50 g/L PP3 demonstrating a reproducible growth profile:

GCFT03b

GCFT05c

GCFT05d

FIG. 55 is a SDS-PAGE analysis showing the time course of GCFT05d (batchfermentation with Proteose Peptone #3), 8% Tris Glycine gel, colloidalstained):

Lane Sample 1 High Molecular Weight Marker 2 Reference, AUXI and AUXII 33 hours 4 4 hours 5 5 hours 6 6 hours 7 7 hours 8 8 hours 9 9 hours 1010 hours  11 11 hours 

FIG. 56 is a SDS-PAGE analysis showing the time course of GCFT05d (batchfermentation with Proteose Peptone #3), (8% Tris Glycine gel, silverstained):

Lane Sample 1 High Molecular Weight Marker 2 Reference, AUXI and AUXII 33 hours 4 4 hours 5 5 hours 6 6 hours 7 7 hours 8 8 hours 9 9 hours 1010 hours  11 11 hours. 

FIG. 57 is a SDS-PAGE analysis showing the time course of DCFT24b(fed-batch fermentation using Phytone peptone), (8% Tris Glycine gel,colloidal stained):

Lane Sample 1 High Molecular Weight Marker 2 AUXI - 0.27 μg 3 AUXII -0.29 μg 4 20 hours - Harvest point 5 19 hours 6 17 hours 7 16 hours 8 15hours 9 14 hours 10 13 hours 11 11.6 hours  12 10.5 hours. 

FIG. 58 illustrates a comparison of growth curves from C. histolyticumfermentations using different lots of PP3:

GCFT05d

GCFT06b

GCFT07c

GCFT07d

GCFT08c

GCFT08d

GCFT09C

GCFT9D.

FIG. 59 illustrates a small scale comparison of three lots of PP3 andevaluation of 100 g/L PP3:

batch 5354796 (50 g/L)

batch 5332398 (50 g/L)

batch 5325635 (50 g/L)

batch 5332398 (100 g/L)

FIG. 60 depicts a growth profiles of two 5 L fermentations utilizing PP3at 100 g/L:

PBFT70c lot #5354796

PBFT70d lot #5325835.

FIG. 61 is a SDS-PAGE analysis of the time course of PBFT70c, 100 g/LPP3 (lot #5354796) fermentation (8% Tris-Glycine):

Lane Sample 1 High Molecular Weight Marker 2 Reference, 0.4 μg AUXI andAUXII 3 3.1 hours 4 4.4 hours 5 5.5 hours 6 6.6 hours 7 8.0 hours 8 9.1hours 9 10.1 hours  10 11.4 hours  11 12.0 hours. 

FIG. 62 is a SDS-PAGE analysis of the timecourse of PBFT70d, 100 g/L PP3(lot #5325635) fermentation (8% Tris-Glycine):

Lane Sample 1 High Molecular Weight Marker 2 Reference, 0.4 μg AUXI andAUXII 3 3.1 hours 4 4.3 hours 5 5.5 hours 6 6.6 hours 7 7.8 hours 8 9.1hours 9 10.1 hours  10 11.3 hours  11 12.0 hours. 

FIG. 63 represents a densitometry analysis of SDS-PAGE to compare cellgrowth to product formation from 5 L fermentation PBFT70c:

Precursor

Aux 1

Aux 2

OD.

FIG. 64 illustrates a Comparison of 100 g/L PP3 process at 5 L and 200 Lscale:

PBFT70c

PBFT70d

200 L scale-up fermentation.

FIG. 65 is a SDS-PAGE analysis of the time course of the 200 Lfermentation (8% Tris-Glycine):

Lane Sample 1 High Molecular Weight Marker 2 AUXI and AUXII mixedreference (1.2 μg) 3 4 hours 4 6 hours 5 8 hours 6 9.4 hours  7 12hours  8 14 hours. 

FIG. 66 represents a densitometry analysis of SDS-PAGE to compare cellgrowth to product formation from 200 L fermentation:

Precursor

Aux 1

Aux 2

OD.

FIG. 67 is a SDS-PAGE analysis of the time course of the 200 Lfermentation (4-12% Bis-Tris):

Lane Sample 1 High Molecular Weight Marker 2 AUXI and AUXII mixedreference (1.2 μg) 3 4 hours 4 6 hours 5 8 hours 6 9.4 hours  7 12hours  8 14 hours. 

FIG. 68 shows a standard curve for densitometry quantification ofcollagenase concentration.

FIG. 69 represents a schematic illustration of the fermentation andharvest of Clostridium histolyticum.

FIGS. 70 (a) and (b) are chromatograms resulting from Hydrophobicinteraction chromatography using Phenyl Sepharose FF (low sub): (a) isfull scale chromatogram and (b) is an expanded chromatogram showingfraction collection.

FIG. 71 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe MUSTANG Q filter step to the TFF 1 step. The gel is stained withColloidal blue and overloaded (2.5 μg total protein/lane) to showcontaminant bands:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 12 2 PostMUSTANG Q filtrate 15 3 Pre HIC Bag 1 15 4 Pre HIC Bag 2 15 5 HICflow-through Bag 1 15 6 HIC flow-through Bag 2 15 7 HIC Peak 1 (0.3M ASwash) 15 8 Post HIC pool (peak 2) 15 9 Pre TFF (post HIC pool + 2 dayhold) 15 10 Post TFF 15 11 Pre Q-AEX (post TFF + overnight hold) 15.

FIG. 72 is an Ion exchange chromatogram (Q Sepharose HP) of the post HICmaterial after concentration and diafiltration into 10 mM Tris, 200 μMleupeptin pH 8.

FIG. 73 is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 1(AUXII) eluted during the ion exchange column (FIG. 5). Gel 1: the gelis stained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 10 2Collagenase ABC I reference 1 μg 15 3 Collagenase ABC II reference 1 μg15 4 Load 1 μg 15 5 Fraction 1 neat 15 6 Fraction 2 neat 15 7 Fraction 3neat 15 8 Fraction 4 {close oversize brace} AUXII 1 μg 15 9 Fraction 5fractions 1 μg 15 10 Fraction 6 1 μg 15 11 Fraction 7 1 μg 15 12Fraction 8 1 μg 15.

FIG. 74 is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 1(AUXII) eluted during the ion exchange column (FIG. 5). Gel 2: the gelis stained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 10 2Collagenase ABC I reference 1 μg 15 3 Collagenase ABC II reference 1 μg15 4 Fraction 9 1 μg 15 5 Fraction 10 1 μg 15 6 Fraction 11 {closeoversize brace} AUXII 1 μg 15 7 Fraction 12 fractions 1 μg 15 8 Peak 1Tail 1 μg 15.

FIG. 75 is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 2(AUXI) eluted during the ion exchange column (FIG. 5). Gel 3: the gel isstained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 10 2Collagenase ABC I reference 1 μg 15 3 Collagenase ABC II reference 1 μg15 4 Fraction 13 neat 15 5 Fraction 14 1 μg 15 6 Fraction 15 1 μg 15 7Fraction 16 AUXI   μg 15 8 Fraction 17 {close oversize brace} fractions  μg 15 9 Fraction 18 1 μg 15 10 Fraction 19 1 μg 15 11 Fraction 20 1 μg15 12 Fraction 21 1 μg 15.

FIG. 76 is a 4-12% Bis Tris SDS-PAGE analysis of fractions from peak 2(AUXI) eluted during the ion exchange column (FIG. 5). Gel 4: the gel isstained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 10 2Collagenase ABC I reference 1 μg 15 3 Collagenase ABC II reference 1 μg15 4 Fraction 22 1 μg 15 5 Fraction 23 1 μg 15 6 Fraction 24 AUXI 1 μg15 7 Fraction 25 {close oversize brace} fractions 1 μg 15 8 Fraction 261 μg 15 9 Fraction 27 1 μg 15 10 Peak 2 Tail 1 μg 15.

FIG. 77 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe anion exchange step to final product. The gel is stained withColloidalblue. Gel 1: 1 μg/lane loading:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 10 2 ABC IReference 15 3 ABC II Reference 15 4 Post IEX AUX I Pool 15 5 Post IEXAUX II Pool 15 6 AUX I Intermediate (DOM: 12 MAY 2006) 15 7 AUX IIIntermediate (DOM: 10 MAY 2006) 15 8 AUX I Intermediate (Pre Mix) 15 9AUX II Intermediate (Pre Mix) 15 10 Drug Substance (DOM: 15 MAY 2006)15.

FIG. 78 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe anion exchange step to final product. The gel is stained withColloidal blue. Gel 2: 2.5 μg/lane loading:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 10 2 ABC IReference 15 3 ABC II Reference 15 4 Post IEX AUX I Pool 15 5 Post IEXAUX II Pool 15 6 AUX I Intermediate (DOM: 12 MAY 2006) 15 7 AUX IIIntermediate (DOM: 10 MAY 2006) 15 8 AUX I Intermediate (Pre Mix) 15 9AUX II Intermediate (Pre Mix) 15 10 Drug Substance (DOM: 15 MAY 2006)15.

FIG. 79 is a SDS-PAGE with 8% Tris Glycine (NB Ref AS/1640/020):

-   -   1. High Molecular Weight Markers    -   2. Blank Lane    -   3. 1 μg Fermentation Filtrate Day 4    -   4. 1 μg Fermentation Filtrate Day 5    -   5. 1 μg Post Mustang Q Day 4    -   6. 1 μg Post HIC Day 3    -   7. 1 μg Post HIC Day 6    -   8. 1 μg Post TFF Day 2    -   9. 1 μg Post TFF Day 4

FIG. 80 is a SDS-PAGE with 8% Tris Glycine:

-   -   1. High Molecular Weight Markers    -   2. 1 μg. AUX-I Reference    -   3. 1 μg AUX-II Reference    -   4. 1 μg Post IEX AUX-I Day 5    -   5. 1 μg Post IEX AUX-I Day 12    -   6. 1 μg Post TEX AUX-II Day 5    -   7. 1 μg Post IEX AUX-II Day 12

FIG. 81 is a SDS-PAGE gel:

-   -   1. High Molecular Weight Markers    -   2. 1 μg AUX-I Reference    -   3. 1 μg AUX-II Reference    -   4. 1 μp AUX-I Intermediate Day 5    -   5. 1 μg AUX-I Intermediate Day 12    -   6. 1 μg AUX-II Intermediate Day 5    -   7. 1 μg AUX-II Intermediate Day 12

FIG. 82 represents analytical chromatography analysis.

FIG. 83 shows protein concentration determination by UV.

FIG. 84 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe 20 L demonstration run-through taken at the point of manufacture andstored at −20° C. The gel is stained with Colloidal blue. 1 μg loading:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 12 2 PostMustang Q filtrate 15 3 Pre HIC Bag 1 15 4 Pre HIC Bag 2 15 5 HICflow-through Bag 1 15 6 HIC flow-through Bag 2 15 7 HIC Peak 1 (0.3M ASwash) 15 8 Post HIC pool 15 9 Pre TFF1 (post HIC pool + weekend hold) 1510 Post TFF 1 15 11 Pre Q-AEX (post TFF + overnight hold) 15.

FIG. 85 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe 20 L demonstration run-through after 22 hrs at Room Temperature. Thegel is stained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 12 2 PreMustang Q filtrate 15 3 Post Mustang Q filtrate 15 4 Pre HIC Bag 1 15 5Pre HIC Bag 2 15 6 Post HIC Pool 15 7 Pre TFF1 {close oversize brace} 1μg loading 15 8 Post TFF1 15 9 Post IEX Aux I Pool 15 10 Post IEX Aux IIPool 15 11 AUX I Intermediate 15 12 AUX II Intermediate 15.

FIG. 86 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe 20 L demonstration run-through after 22 hrs at 37° C. The gel isstained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 12 2 PreMustang Q filtrate 15 3 Post Mustang Q filtrate 15 4 Pre HIC Bag 1 15 5Pre HIC Bag 2 15 6 Post HIC Pool 15 7 Pre TFF1 {close oversize brace} 1μg loading 15 8 Post TFF1 15 9 Post IEX AUX I Pool 15 10 Post IEX AUX IIPool 15 11 AUX I Intermediate 15 12 AUX II Intermediate 15.

FIG. 87 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe 20 L demonstration run-through after 94 hrs at Room Temperature. Thegel is stained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 12 2 PreMustang Q filtrate 15 3 Post Mustang Q filtrate 15 4 Pre HIC Bag 1 15 5Pre HIC Bag 2 15 6 Post HIC Pool 15 7 Pre TFF1 {close oversize brace} 1μg loading 15 8 Post TFF1 15 9 Post IEX Aux I Pool 15 10 Post IEX Aux IIPool 15 11 AUX I Intermediate 15 12 AUX II Intermediate 15.

FIG. 88 is a 4-12% Bis-Tris SDS-PAGE analysis of in-process samples fromthe 20 L demonstration run-through after 94 hrs at 37° C. The gel isstained with Colloidal blue:

Load Lane Sample volume, μl 1 Mark 12 Molecular Weight Marker 12 2 PreMustang Q filtrate 15 3 Post Mustang Q filtrate 15 4 Pre HIC Bag 1 15 5Pre HIC Bag 2 15 6 Post HIC Pool 15 7 Pre TFF1 {close oversize brace} 1μg loading 15 8 Post TFF1 15 9 Post IEX Aux I Pool 15 10 Post IEX Aux IIPool 15 11 AUX I Intermediate 15 12 AUX II Intermediate 15.

FIG. 89 is an 8% Tris-Glycine SDS-PAGE analysis of selected post IEX AUXI and post IEX AUX II fractions. Fractions were selected from the 20 Ldemonstration run which were enriched for the required contaminantprotein. The gel is stained with Colloidal blue:

Load Lane Sample volume, μl 1 Post IEX AUX I Fraction 16 20 2 Post IEXAUX I Fraction 16 20 3 Post IEX AUX I Fraction 16 {close oversize brace}eluted with 20 4 Post IEX AUX I Fraction 16 AUXI 20 5 Post IEX AUX IFraction 16 20 6 Post IEX AUX II Fraction 2 20 7 Post IEX AUX IIFraction 2 20 8 Post IEX AUX II Fraction 2 {close oversize brace} elutedwith 20 9 Post IEX AUX II Fraction 2 AUXII 20 10 Post IEX AUX IIFraction 2 20.

FIG. 90 is an 8% Tris-Glycine SDS-PAGE analysis of selected post IEX AUXI and post IEX AUX II fractions. Fractions were selected from purifiedmaterial generated from fermentation 20 L PP3 and enriched for the ˜90kDa contaminant protein. The gel is stained with Colloidal blue:

Load Lane Sample volume, μl 1 High Molecular Weight marker 20 2 Post IEXFraction B7 R2 20 3 Post IEX Fraction B7R2 eluted with 20 4 Post IEXFraction B7R2 {close oversize brace} AUXI 20 5 Post IEX Fraction B7R2 206 Post IEX Fraction D1 20 7 Post IEX Fraction D1 20 8 Post IEX FractionD1 {close oversize brace} eluted with 20 9 Post IEX Fraction D1 AUXII 2010 Post IEX Fraction D1 20.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel collagenase drug substance comprising amixture of highly purified collagenase I and collagenase II in a massratio of about 1 to 1. It has been discovered that a compositioncomprising a mixture of collagenase I and collagenase II in anartificial mass ratio of 1 to 1 provides highly reproducible and optimalenzymatic activity and imparts superior therapeutic effect whilelowering the potential for side effects. It is understood that the terms“drug substance”, “drug product” or “collagenase composition” can beused interchangeably.

In one embodiment, the present invention provides a drug substanceconsisting of collagenase I and collagenase II having the sequence ofClostridium histolyticum collagenase I and collagenase II, respectively,having a mass ratio of about 1 to 1 with a purity of at least 95% byarea, and preferably a purity of at least 98% by area.

In another embodiment, the present invention provides a drug substance,wherein the drug substance having at least one specification selectedfrom table A below:

TABLE A Specification Test AUX-I AUX-II Appearance Clear colourless andessentially free from particulate matter Potentiometric Measure of 7.5to 8.5 pH of Solution Endotoxin   <10 EU/mL Identity (and purity) byMajor collagenase band Major collagenase band SDS-PAGE (Reduced between98-188 kDa between 97-200 kDa; conditions, Coomasie and MW markers MWmarkers; major bands silver stained) comparable to reference standardTotal Protein by 0.8-1.2 mg/mL   Absorbance Spectroscopy SRC assay(AUX-I) 13 000-23 000 fSRC NA units/mg GPA assay (AUX-II) NA 230 000-430000 fGPA units/mg Residual host cell protein Comparable to referencestandard; no individual impurity band exhibiting greater intensity than1% BSA intensity marker Residual host cell DNA  ≦10 pg/dose Analysis ofProteins using the ≧98% main peak; ≦2% aggregates by area Agilent 1100HPLC System (Aggregation by size exclusion chromatography) Analysis ofProteins using 2 major peaks (AUX I & AUX II), combined ≧97% by theAgilent 1100 HPLC area; Retention times of AUX-I and AUX-II within 5%System (Identity and purity of reference by reverse phase liquidchromatography) Analysis of Proteins using ≦1% by area the Agilent 1100HPLC System (Residual clostripain by reverse phase liquid chromatographyAnalysis of Proteins using ≦1% by area the Agilent 1100 HPLC System(Residual gelatinase by anion exchange chromatography) Residualleupeptin by    ≦1 ug/mg w/w reverse phase chromatography Bioburden   <1cfu/mL

In one aspect, the invention provides a process for producing a drugsubstance consisting of collagenase I and collagenase II having thesequence of Clostridium histolyticum collagenase I and collagenase II,respectively, having a mass ratio of about 1 to 1 with a purity of atleast 95% by area, comprising the steps of:

-   -   a) fermenting Clostridium histolyticum;    -   b) harvesting a crude product comprising collagenase I and        collagenase II;    -   c) purifying collagenase I and collagenase II from the crude        harvest via filtration and column chromatography; and    -   d) combining the collagenase I and collagenase II purified from        step (c) at a ratio of about 1 to 1.

In one preferred embodiment, the fermentation step is conducted in thepresence of a porcine derived, a phytone peptone or a vegetable peptonemedium. More preferably, the porcine derived medium is proteose peptone#3.

In one embodiment, the invention provides a fermentation procedurecomprising the steps of:

-   -   a) inoculating the medium in a first stage;        -   with Clostridium histolyticum and agitating the mixture;    -   b) incubating the mixture from step (a) to obtain an aliquot;    -   c) inoculating the medium in a second stage with aliquots        resulting from step (b) and agitating the mixture;    -   d) incubating mixtures from step (c);    -   e) inoculating the medium in a third stage with aliquots        resulting from step (d) and agitating;    -   f) incubating mixtures from step (e);    -   g) inoculating the medium in a fourth stage with an aliquot        resulting from step (f) and agitating;    -   h) incubating mixtures from step (g); and    -   i) harvesting culture resulting from step (h) by filtration.        In a preferred embodiment, the fermentation procedure comprises        the steps of:

a) Inoculating 3×25 mL PP3 (proteose peptone) medium with 3×250 μL ofWCB (25 mL cultures in 3×125 mL shake flasks, contained within Anaerobegas jar) at a temperature set point of 37° C., and agitating the mixtureat 125 rpm;

-   -   b) incubating the mixture from step (a) for 12 hours;    -   c) inoculating Inoculate 4×200 mL PP3 medium with 4×5 mL        aliquots from 1 of the above 25 mL cultures (200 mL cultures in        4×500 mL shake flasks, contained within Anaerobe gas jar) at a        temperature set point of 37° C., and agitating the mixture at        125 rpm;    -   d) incubating mixtures from step (c) for 12 hours;    -   e) inoculating 14.4 L of PP3 medium with 3×200 mL culture (15 L        culture in 20 L fermenter) at a temperature set point of 37° C.        and pH set point of 7.00, and agitating the mixture at 125 rpm;    -   f) incubating mixtures from step (e) for 12 hours;    -   g) inoculating 192 L of PP3 medium with 8 L of 15 L culture (200        L culture in 270 L fermenter) at a temperature set point of        37° C. and pH set point of 7.00, and agitating the mixture at        125 rpm;    -   h) incubating mixtures from step (g) for 14 hours; and    -   i) harvesting 200 L culture by filtration (depth followed by 0.2        μm) via Millipore Millistak 4 m² and 0.2 μm filter (2×Millipore        Express XL 10 filters) at a flow rate of 200 L/h.

In one embodiment, the invention provides a purification procedurecomprising the steps of:

-   -   a) filtering the crude harvest through a MUSTANG Q        anion-exchange capsule filter;    -   b) adding ammonium sulphate; preferably to a final concentration        of 1M;    -   c) filtering the crude harvest; preferably through a 0.45 μm        filter;    -   d) subjecting the filtrate through a HIC column; preferably a        phenyl sepharose 6FF (low sub);    -   e) adding leupeptin to the filtrate; preferably to a final        concentration of 0.2 mM to post HIC eluted product;    -   f) removing the ammonium sulfate and maintaining leupeptin for        correct binding of collagenase I and collagenase II with buffer        exchange by TFF;    -   preferably with buffer exchange by TFF;    -   g) filtering the mixture of step (f); preferably through a 0.45        μm filter;    -   h) separating collagenase I and collagenase II using Q-Sepharose        HP;    -   i) preparing TFF concentration and formulation for collagenase I        and collagenase II separately; wherein TFF is a tangential flow        filtration using 10 and/or 30K MWCO (molecular weight cut-off)        PES or RC-polyethersulfone or regenerated cellulose filter        membranes. Provides means to retain and concentrate select        protein and exchange the protein from one buffer solution into        another; and    -   j) filtering through a 0.2 μm filtration system.

The drug substance of the present invention includes both collagenase Iand collagenase II. A preferred source of crude collagenase is from abacterial fermentation process, specifically the fermentation of C.histolyticum (C. his). In one embodiment of the invention, afermentation process is described. The crude collagenase obtained fromC. his may be purified by a variety of methods known to those skilled inthe art, including dye ligand affinity chromatography, heparin affinitychromatography, ammonium sulfate precipitation, hydroxylapatitechromatography, size exclusion chromatography, ion exchangechromatography, and metal chelation chromatography. Crude and partiallypurified collagenase is commercially available from many sourcesincluding Advance Biofactures Corp., Lynbrook, N.Y.

Both collagenase I and collagenase II are metalloproteases and requiretightly bound zinc and loosely bound calcium for their activity (EddieL. Angleton and H. E. Van Wart, Biochemistry 1988, 27, 7406-7412). Bothcollagenases have broad specificity toward all types of collagen(Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260 p 2771-2776).Collagenase I and Collagenase II digest collagen by hydrolyzing thetriple-helical region of collagen under physiological conditions(Steinbrink, D; Bond, M and Van Wart, H; (1985), JBC, 260 p 2771-2776).Even though each collagenase shows different specificity (e.g. each havea different preferred amino sequence for cleavage), together, they havesynergistic activity toward collagen [Mandl, I., (1964), Biochemistry,3: p. 1737-1741; Vos-Scheperkeuter, G H, (1997), Cell Transplantation,6: p. 403-412]. Collagenase II has a higher activity towards all kindsof synthetic peptide substrates than collagenase I as reported for classII and class I collagenase in the literatures. [Bond, M. D. (1984),Biochemistry, 23: p. 3085-3091. Hesse, F, (1995), TransplantationProceedings, 27: p. 328′7-3289].

Examples of collagen mediated-diseases that may be treated by thecompositions and methods of the invention include but are not limitedto: Dupuytren's disease; Peyronie's disease; frozen shoulder (adhesivecapsulitis), keloids; hypertrophic scars; depressed scars such as thoseresulting from inflammatory acne; post-surgical adhesions; acnevulgaris; lipomas, and disfiguring conditions such as wrinkling,cellulite formation and neoplastic fibrosis. U.S. Pat. Nos. 6,086,872and 5,589,171 incorporated herein by reference disclose the use ofcollagenase preparations in the treatment of Dupuytren's disease. U.S.Pat. No. 6,022,539 incorporated herein by reference discloses the use ofcollagenase preparations in the treatment of Peyronie's disease.

In addition its use in treating collagen-mediated diseases, thecomposition of the invention is also useful for the dissociation oftissue into individual cells and cell clusters as is useful in a widevariety of laboratory, diagnostic and therapeutic applications. Theseapplications involve the isolation of many types of cells for varioususes, including microvascular endothelial cells for small diametersynthetic vascular graft seeding, hepatocytes for gene therapy, drugtoxicology screening and extracorporeal liver assist devices,chondrocytes for cartilage regeneration, and islets of Langerhans forthe treatment of insulin-dependent diabetes mellitus. Enzyme treatmentworks to fragment extracellular matrix proteins and proteins whichmaintain cell-to-cell contact. Since collagen is the principle proteincomponent of tissue ultrastructure, the enzyme collagenase has beenfrequently used to accomplish the desired tissue disintegration. Ingeneral, the composition of the present invention is useful for anyapplication where the removal of cells or the modification of anextracellular matrix, are desired.

Collagenase compositions of the invention may also be prepared by mixingeither a specific number of activity units or specific masses of thepreferably purified enzymes. Collagenase activity can be measured by theenzyme's ability to hydrolyze either synthetic peptide or collagensubstrate. Those skilled in the art will recognize that enzyme assaysother than those disclosed herein may also be used to define and preparefunctionally equivalent enzyme compositions.

Another aspect of the present invention is the reproducible optimizationof the 1 to 1 mass ratio of collagenase I to collagenase II in thecomposition of the invention. The reproducibility of the ratio ofcollagenase I to collagenase II has previously been a challenge becauseof several factors. First, commercial fermentation of Clostridiumgenerally results in a 1 to 2 ratio of collagenase I and collagenase II.Second, the purification procedures are known to alter this ratiosignificantly resulting in inconsistent ratios of purified product. Theoptimized fixed mass ratio of the composition of the present inventionmaximizes the synergistic activity provided by the two differentcollagenases resulting in superior therapeutic benefit.

The invention also provides pharmaceutical formulations of thecompositions of the invention. The pharmaceutical formulations of thepresent invention comprise a therapeutically effective amount of acollagenase composition of the present invention formulated togetherwith one or more pharmaceutically acceptable carriers or excipients.

As used herein, the term “pharmaceutically acceptable carrier orexcipient” means a non-toxic, inert solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.Some examples of materials which can serve as pharmaceuticallyacceptable carriers are sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; cellulose and itsderivatives such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; glycolssuch as propylene glycol; esters such as ethyl oleate and ethyl laurate;agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, perfuming agents, preservatives and antioxidants canalso be present in the composition, according to the judgment of theformulator.

The pharmaceutical compositions of this invention may be administeredparenterally, topically, or via an implanted reservoir. The termparenteral as used herein includes subcutaneous, intracutaneous,intravenous, intramuscular, intraarticular, intraarterial,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. In a preferred embodiment, thecomposition is injected into the disfiguring tissue. In the case ofPeyronie's or Duputyren's diseases or adhesive capsulitis, thecomposition is injected into the cord or plaque. The term “localadministration” is defined herein to embrace such direct injection.

Furthermore, particularly good results can be obtained by immobilizingthe site of injection after administration. For example, the site ofadministration can be immobilized for 4 or more hours.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1, 3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use. The sterile solutions may also be lyophilized forlater use.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

In one preferred embodiment, the drug substance of the invention is alyophilized injectable composition formulated with lactose. In oneembodiment each milligram of injectable collagenase is formulated with1.9 mg of lactose. In another embodiment, each milligram of injectioncollagenase preferably has approximately 2800 SRC units and 51000 unitsmeasured with a potency assay using a synthetic substrate, pzGPGGPA.

In another preferred embodiment, the collagenase composition of theinvention is a lyophilized injectable composition formulated withSucrose, Tris at a pH level of about 8.0. Most preferably, 1.0 mg of thedrug substance of the invention is formulated in 60 mM Sucrose, 10 mMTris, at a pH of about 8.0 (this equates to 20.5 mg/mL of sucrose and1.21 mg/mL of Tris in the formulation buffer). Examples of some of theformulations include, but not limited to: for a 0.58 mg of the drugsubstance dose, 18.5 mg of sucrose and 1.1 mg of Tris are added in eachvial, where the targeting a vial fill volume is 0.9 mL; and for a 0.58mg of the drug substance dose, 12.0 mg sucrose (multicompendial) and 0.7mg of Tris (multicompendial).

In accordance with the invention, methods are provided for treatingcollagen-mediated diseases comprising the step of administering to apatient in need thereof, a therapeutically effective amount of acomposition of the invention, or a therapeutically effective amount of apharmaceutical formulation of the invention. By a “therapeuticallyeffective amount” of a compound of the invention is meant an amount ofthe compound which confers a therapeutic effect on the treated subject,at a reasonable benefit/risk ratio applicable to any medical treatment.

The therapeutic effect may be objective (i.e., measurable by some testor marker) or subjective (i.e., subject gives an indication of or feelsan effect). Effective doses will also vary depending on route ofadministration, as well as the possibility of co-usage with otheragents. It will be understood, however, that the total daily usage ofthe compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific therapeutically effective dose level for any particular patientwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or contemporaneously with the specific compound employed;and like factors well known in the medical arts.

The drug substance for injectable collagenase consists of two microbialcollagenases, referred to as Collagenase AUX I and Collagenase ABC I andCollagenase AUX II and Collagenase ABC II. It is understood that theterms “Collagenase I”, “ABC I”, “AUX I”, “collagenase AUX I”, and“collagenase ABC I” mean the same and can be used interchangeably.Similarly, the terms “Collagenase II”, “ABC II”, “AUX II”, “collagenaseAUX II”, and “collagenase ABC II” refer to the same enzyme and can alsobe used interchangeably. These collagenases are secreted by bacterialcells. They are isolated and purified from Clostridium histolyticumculture supernatant by chromatographic methods. Both collagenases arespecial proteases and share the same EC number (E.C 3.4.24.3).

Collagenase AUX I has a single polypeptide chain consisting ofapproximately 1000 amino acids with a molecular weight of 115 kDa.Collagenase AUX II has also a single polypeptide chain consisting ofabout 1000 amino acids with a molecular weight of 110 kDa.

Even though the literature indicates that there are sequence homologiesin regions of collagenase AUX I and AUX II, the two polypeptides do notseem to be immunologically cross reactive as indicated by the westernblot analysis.

The drug substance (collagenase concentrate) has an approximately 1 to 1mass ratio for collagenase AUX I and AUX II. The collagenase concentratehas an extinction coefficient of 1.528.

All references cited herein, whether in print, electronic, computerreadable storage media or other form, are expressly incorporated byreference in their entirety, including but not limited to, abstracts,articles, journals, publications, texts, treatises, internet web sites,databases, patents, and patent publications.

EXAMPLES

The compositions and processes of the present invention will be betterunderstood in connection with the following examples, which are intendedas an illustration only and not limiting of the scope of the invention.Various changes and modifications to the disclosed embodiments will beapparent to those skilled in the art and such changes and modificationsincluding, without limitation, those relating to the processes,formulations and/or methods of the invention may be made withoutdeparting from the spirit of the invention and the scope of the appendedclaims.

Process 2: Fermentation Process

This work was set out to develop a fermentation process that aimed atdelivering a target yield of 250 mg/L of total collagenases ABC I & IIfrom the 5 L fermentation scale process in an animal free componentgrowth media. Various potential alternative nitrogen sources werescreened to see if they had any affect on collagenase expression overand the above the phytone component currently used in the growth media.An experiment comparing productivities from two strains of C.histolyticum, 004 and 013, was to determine any differences between thetwo strains with respect to growth kinetics, collagenase productivityand production of contaminating proteases grown in an animal derivedmedia. This comparison highlighted significant differences betweengrowing the C. histolyticum strain in animal derived media as opposed toanimal free growth media.

Previous results described that increased concentrations of phytone andyeast extract were shown to support higher biomass concentrations andhence higher levels of total collagenase expression. In an attempt tofurther increase biomass concentrations and total collagenaseproductivity of the optimised batch fermentation media, a fed-batchfermentation strategy was designed. Two 5 L fermentations wereperformed, one with a high concentration of media in the batch phasefollowed by a low concentration feeding phase, the second with a lowconcentration of media in the batch phase followed with a highconcentration feeding phase. Both fermentations produced high biomassconcentrations, however the high concentration batch phase showedrelatively low levels of collagenase expression. The low concentrationbatch fermentation showed very high levels of collagenase expression(˜280 mg/L), however this culture also produced significant quantitiesof the contaminating protease, clostripain.

Although the low concentration batch fermentation gave very good resultswith respect to expression of the collagenases, the highly concentratedphytone and yeast extract feed solution was very difficult to prepare.Two additional fermentations were performed, the first was a repeat ofthe previous successful fed-batch fermentation the second had a slightlyhigher concentration batch phase media composition with a lowerconcentrated feeding solution. Both fermentations achieved similarbiomass concentrations and showed the same expression profile of thecollagenases and clostripain. The quantity of collagenase produced wasagain estimated at approximately 280 mg/L in both fermentations.However, these fermentations produced significant quantities of thecontaminating protease clostripain.

A selection of alternative nitrogen sources were assessed for theirability to replace the phytone peptone used in the fed-batchfermentation strategy. The C. histolyticum grew extremely well on thevegetable peptones reaching optical densities (600 nm) of 4 to 5 units.However, SDS-PAGE analysis of these fermentations showed no expressionof either collagenase or clostripain. Due to the luxuriant cell growthobserved on these peptones it was thought that the concentration ofcomplex nitrogen source was too high resulting in an inhibition ofprotease expression. A second set of fermentations was therefore carriedout using the alternative peptones at 50 g/L in a batch strategy. Whenthe fermentations were analyzed by SDS-PAGE no expression of collagenaseor clostripain was seen again. A fed-batch fermentation using phytonepeptone was supplemented with three amino acids, glutamine, tryptophanand asparagine. These amino acids were identified as being present inlower amounts in the non-animal media. The growth profile of thefermentation was very similar to that of the fed-batch fermentationwithout amino acid supplementation. SDS-PAGE analysis showed a similaryield of collagenase but a slightly lower level of clostripain. Theclostripain assay showed reduced activity in the amino supplemented whencompared to the control fed-batch fermentation. The reduction inclostripain activity whilst still significant was not as great as thedifference between animal and non-animal media.

The assessment of the primary recovery step of the collagenases usingammonium sulphate precipitation was carried out on 0.2 μm filtrates ofthe crude fermentation supernatants. The aim here was to help increasethe collagenase yield and ideally decrease the quantity of clostripainthat was carried through the process. Initially ammonium sulphateconcentrations of 100-400 g/L were assessed. Ammonium sulphate at 400g/L resulted in significant recovery of collagenase. A further study wascarried out with a higher range of ammonium sulphate (400-520 g/L). Inaddition, the effect of decreasing the pH to 6.0 and oxygenating themedia prior to precipitation were also investigated. No difference wasobserved in either the quantity of the collagenases or clostripainrecovered from the supernatant under any of these conditions. The pelletgenerated from 400 g/L ammonium sulphate was the easiest to resuspend.

The study to compare the two strains of C. histolyticum (004 and 013)showed that the productivity of the collagenases from the animal derivedmedia was lower than that of the optimal non-animal derived media.SDS-PAGE analysis, supported by an enzymatic assay for clostripainactivity, highlighted that there were significantly lower quantities ofclostripain in the material produced from the animal derived media thanthe non-animal media. This highlighted the fact that the feedstockproduced from the non-animal derived media fermentation was asignificantly different feedstock material from the fermentation usinganimal derived media with respect to the production of contaminatingproteases.

1^(st) Set of Fed-Batch Fermentations—DCFT24

The results from the process development work showed that the use of anenriched media (100 g/L phytone peptone and 50 g/L yeast extract)resulted in the expression of higher amounts of collagenases compared tothe original media (50 g/L phytone peptone and 8.5 g/L yeast extract).In addition, it initially appeared that it reduced the amounts ofclostripain produced.

Two 5 L fermentations were then performed. Firstly the strategyconsisted of a long batch phase/short fed-batch phase, whereas thesecond consisted of a short batch phase/long fed-batch phase. In bothstrategies at the end of the fermentation (after 20 h) theconcentrations of phytone peptone and yeast extract were 100 g/L and 50g/L, respectively, as in the case of the batch fermentations. Table 1and 2 detail the media recipes and strategies used.

TABLE 1 Media recipe and fed-batch strategy Long batch - short fed-batchConcentrations at harvest Component Batch phase Feed point PhytonePeptone 100 g/L 100 g/L 100 g/L Yeast extract 50 g/L 50 g/L 50 g/LGlucose 10 g/L 10 g/L 10 g/L Filtered sterilised KH₂PO₄ 1.92 g/L K₂HPO₄1.25 g/L Na₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/L Vitaminsolution 10 mL/L Volume 4 L 1 L ~5 L

TABLE 2 Media recipe and fed-batch strategy Short batch - long fed-batchConcentrations at harvest Component Batch phase Feed point PhytonePeptone 40 g/L 254 g/L 100 g/L Yeast extract 10 g/L 153 g/L 50 g/LGlucose 7.5 g/L 17.8 g/L 10 g/L Filtered sterilised KH₂PO₄ 1.92 g/LK₂HPO₄ 1.25 g/L Na₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/L Vitaminsolution 10 mL/L Volume 4 L 1 L ~5 L

FIG. 1 shows the growth curves (OD_(600 nm) vs time) from the twofermentations, whereas FIG. 2 shows the net growth curves (NetOD_(600 nm) vs time). It was observed that the cells from the firstfermentation grew very fast and reached their maximum OD afterapproximately 10 hours. This was due to the fact that the media in thebatch phase was very rich. During the fed-batch phase the cells did notappear to grow. The OD values decreased slightly, which could be partlyattributed to the fact that the cells were dying and to the dilutioneffect of the feed in to the fermenter.

For the second fermentation, the fed-batch phase was started after 6hours. At that point the OD value would have been low, as suggested bythe growth curve in FIG. 1. The cells continued to grow slowly up toapproximately 18 hours.

It was noted that the net growth curves in FIG. 2 suggested that thecell densities in DCFT24b fermentation were higher than in DCFT24afermentation. The OD_(600 nm) of the media prior to inoculation wasapproximately 1.7, whereas in DCFT24b it was approximately 0.4. Thesedifferences are due to the fact when the fermenters are autoclaved aprecipitate is formed. For DCFT24a, higher amounts were formed comparedto DCFT24b.

SDS PAGE Gels:

SDS PAGE analysis (8% Tris-Glycine gels) of the supernatant samples werecarried out for each for the two fermentations. The gels are shown inFIGS. 3 and 4. A semi-quantitative SDS PAGE gel was also produced forthe harvest point sample of the second fermentation.

The SDS PAGE gel analysis in FIG. 4 indicated that very low amounts ofthe collagenases were expressed. This could be due to the fact that thecells grew very fast during the batch phase and as a result the maximumcell concentration was reached after approximately 10 hours. Incontrast, very high level of collagenase expression was observed in thesecond fermentation, probably due to the fact that the cells grew moreslowly during the short batch phase and continued to grow during thefed-batch phase. Thus the invention relates to an improved fermentationmethod for C. his wherein cell growth is controlled and slow during theshort batch phase and continuing growth during the fed-batch phase. Slowgrowth is defined to mean that the rate of growth during the short batchphase does not result in a maximum cell concentration prior to thefed-batch phase, such as within about 10 hours of the beginning of thefermentation process. In a preferred embodiment, the rate of growth isapproximately that resulting from the second fermentation cycledescribed herein.

Estimated collagenase productivities from the semi-quantitative SDS PAGEgel at the harvest point of the second fermentation cycle (FIG. 5), were132 mg/L for collagenase ABC I and 158 mg/L for collagenase ABC II.Comparing these values with those previously obtained, there isapproximately a 3-fold increase in the expression levels using thefed-batch strategy.

The next step was to perform an additional set of fed-batchfermentations using slightly modified fed-batch strategies and media.The aim was to improve the scalability and robustness of thefermentation process.

The media recipe for this fermentation was the same as above, with theexception that the phytone peptone and the yeast extract in the batchphase were filter sterilised instead of being autoclaved. This was donein order to avoid autoclaving the yeast extract and phytone, which canpotentially affect their composition by heat and denaturation ofproteins in the media. For fermentation DCFT26b, the amount of yeastextract and phytone peptone was increased. This was done so that theconcentration of yeast extract and peptone in the feed was less thanthat in DCFT26a and thus easier to make up and filter sterilise. Forboth fermentations the strategy followed was the same, a 6 h batch phasefollowed by a 14 h fed-batch phase. Tables 3 and 4 present the mediarecipes, whereas FIG. 6 the strategy used for both fermentations.

TABLE 3 Media recipe and fed-batch strategy for DCFT26a DCFT26a - (shortbatch - long fed-batch) Concentrations at Component Batch phase Feedharvest point Phytone Peptone 40 g/L 254 g/L 100 g/L Yeast extract 10g/L 153 g/L 50 g/L Glucose 7.5 g/L 17.8 g/L 10 g/L Filtered sterilisedKH₂PO₄ 1.92 g/L K₂HPO₄ 1.25 g/L Na₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium0.08 g/L Vitamin solution 10 mL/L Volume 3.6 L 1.4 L ~5 L

TABLE 4 Media recipe and fed-batch strategy for DCFT26b DCFT26b - (shortbatch - long fed-batch) Concentrations at Component Batch phase Feedharvest point Phytone Peptone 60 g/L 151.4 g/L 100 g/L Yeast extract 20g/L 127.1 g/L 50 g/L Glucose 7.5 g/L 17.8 g/L 10 g/L Filtered sterilizedKH₂PO₄ 1.92 g/L K₂HPO₄ 1.25 g/L Na₂PO₄ 3.5 g/L NaCl 2.5 g/L Magnesium0.08 g/L Vitamin solution 10 mL/L Volume 3.6 L 1.4 L ~5 L

FIG. 7 shows the growth curves (OD_(600 nm) vs time) from the twofermentations, whereas FIG. 8 shows the net growth curves (NetOD_(600 nm) vs time).

The growth curves for DCFT26a and DCFT26b were very similar to that ofDCFT24b shown in FIG. 2. The cells grew slowly during the fed-batchphase and reached a final net OD_(600 nm) of approximately 3.5.

SDS PAGE Gels of Fermentation Samples:

SDS PAGE analysis (8% Tris-Glycine gels) of the supernatant samples wascarried out for each of the two fermentations (FIG. 9 and FIG. 10). Inaddition, in order to have a better estimate of the amount ofcollagenases, a semi-quantitative SDS PAGE gel was conducted for theharvest sample point of DCFT26a (FIG. 11) and DCFT26b (FIG. 12).

In both fermentations the levels of collagenases were similar to thosein DCFT24b (FIG. 3). The semi-quantitative SDS PAGE gel shows that verysimilar levels to DCFT24b (between 280 mg/L to 300 mg/L totalcollagenase) were obtained for both DCFT26a and DCFT26b. The harvestpoint of the DCFT26a fermentation cycle (FIG. 11) were ˜142 mg/L forcollagenase I and ˜132 mg/L for collagenase II. The harvest point of theDCFT26b fermentation cycle (FIG. 12) were ˜147 mg/L for collagenase Iand ˜158 mg/L for collagenase II. The levels of clostripain, as in thecase of DCFT24b, were still high.

Study of the Ammonium Sulphate Precipitation Step:

The results from these fermentations indicated that although the levelsof collagenases were high using the fed-batch strategy, the levels ofclostripain were also still significantly high. Therefore, a small scaleexperimental study was set up to investigate the effect of the ammoniumsulphate concentration on the recovered amounts of clostripain andcollagenases in the precipitated pellet from the filtered fermentationsupernatant.

In order to evaluate the efficiency of the ammonium sulphateprecipitation step, 6×100 mL supernatant samples were harvested fromfermentation DCFT26a. These samples were precipitated with 6 differentammonium sulphate concentrations as detailed in the following table. Thepellets were re-suspended in 3.3 mL of WFI and dialysed against 100 mMof K₂HPO₄ (pH 6.7).

TABLE 5 Ammonium sulphate concentrations that were used to precipitate100 mL supernatant samples from DCFT26a. Ammonium sulphate Percentagesaturation Concentration (g/L) 15% 100 g/L 22.5%  150 g/L 30% 200 g/L37.5%  250 g/L 45% 300 g/L 60% 400 g/LThe post-dialysed samples were then analysed by SDS PAGE analysis.FIG. 13: post-dialysed harvest point sample precipitated with 15% and22.5%.FIG. 14: post-dialysed harvest point sample precipitated with 30% and37.5%.FIG. 15: post-dialysed harvest point sample precipitated with 45% and60%.

The gels show that in the case where the ammonium sulphate used wasbetween 15% to 45% saturation, the levels of collagenases in thepost-dialysed samples were very low. The recovery in these cases seemedto be less than 5%.

In the case where 60% saturation of ammonium sulphate was used (400 g/L)the levels of collagenases in the post-dialysed sample were very high(FIG. 15). By comparing the intensity of the bands (sample versusreferences) it can be estimated that approximately 70 mg/L for each ofthe collagenases were present in the post-dialysed sample. This suggestsa recovery of about 50 to 60%, since according to thesemi-quantification gel for DCFT26a (FIG. 11) there were approximately130 mg/L of each of the collagenases in the harvest point sample.

Thus, the invention relates to the use of the media recipe (of course,amounts set forth therein are approximated) set forth above in DCFT26band the use of ammonium sulphate to precipitate collagenase whereinabout 400 g/liter of ammonium sulfate is added to thecollagenase-containing medium.

3^(rd) Set of Fed-Batch Fermentations

Here the primary aim was to assess the reproducibility of the developedfed-batch strategy. A fed-batch fermentation was performed which was areplicate fermentation of DCFT26b. In addition, the ammoniumsulphate/precipitation steps were investigated in more detail comparedto the previous small-scale study. More specifically, the aim was toexamine the effect of various ammonium sulphate concentrations, from 60%(400 g/L) up to 80% (530 g/L) on the recovery of collagenases andclostripain in the post precipitated/dialysed samples. In addition, twomethods of treating the harvested supernatant samples were alsoassessed, i.e., shifting the pH and oxygenating the media.

Growth Curve:

The media and fed-batch strategy used was exactly the same as DCFT26b.FIG. 16 shows the growth curve (OD_(600 nm) vs time) and the net growthcurve (Net OD_(600 nm) vs time) from the fermentation. The growth curvewas very similar to that of DCFT26b, indicating the good reproducibilityof the process.

SDS PAGE analysis (8% Tris-Glycine gels) of the supernatant samplestaken throughout the fermentation indicated that the levels ofcollagenases and clostripain were very similar to those of DCFT26b (SDSPAGE gel not shown). A semi-quantitative SDS PAGE gel (8% Tris-Glycinegel) was performed for the harvest point sample (FIG. 17). The gelsuggests that there is higher than 120 mg/L of each of the collagenasespresent, similar to the levels observed in DCFT26b.

Ammonium Sulfate Precipitation of Fermentation Harvest Samples:

In order to evaluate the efficiency of the ammonium sulphateprecipitation step, 7×500 mL supernatant samples were harvested. Thesewere precipitated using the following six methods.

In all cases, the pellets were re-suspended in 16.5 mL of WFI anddialysed against 100 mM of K₂HPO₄ (pH 6.7), with the exception of method4, where the pellet was re-suspended in 16.5 mL of 100 mM of K₂HPO₄ (pH6) and dialysed against the same buffer.

SDS PAGE gels were then performed in order to estimate the amounts ofcollagenases in the post-dialysed samples and evaluate the recovery ofthe precipitation/dialysis steps. The methods for precipitation/dialysisfollowed are the following:

-   1 Precipitation with 400 g/L of ammonium sulphate added all at once    into the supernatant sample. Dialysis against 100 mM of K₂HPO₄, pH    6.7.-   2 Precipitation with 400 g/L of ammonium sulphate added slowly    (about 30 min) into the supernatant sample. Dialysis against 100 mM    of K₂HPO₄, pH 6.7.-   3 Precipitation with 400 g/L of ammonium sulphate added slowly    (about 30 min) into the supernatant sample, which was    pre-oxygenated. This was done by oxygenating for approximately 10    minutes 500 mL of cell culture harvested from the fermenter. The    culture was then filter sterilised. The pellet formed after ammonium    sulphate precipitation was dialysed against 100 mM of K₂HPO₄ pH 6.7.-   4 Precipitation with 400 g/L of ammonium sulphate added slowly    (about 30 min) into the supernatant sample, the pH of which was    changed to pH 6 by adding 5N HCl. The pellet formed after was    dialysed against 100 mM of K₂HPO₄, pH 6.-   5 Precipitation with 440 g/L of ammonium sulphate added slowly    (about 30 min) into the supernatant sample. Dialysis against 100 mM    of K₂HPO₄, pH 6.7.-   6 Precipitation with 480 g/L of ammonium sulphate added slowly    (about 30 min) into the supernatant sample. Dialysis against 100 mM    of K₂HPO₄, pH 6.7.-   7 Precipitation with 520 g/L of ammonium sulphate added slowly    (about 30 min) into the supernatant sample. Dialysis against 100 mM    of K₂HPO₄, pH 6.7.

The ammonium sulphate did not completely dissolve when added at 480 g/Land 520 g/L in the supernatant samples, whereas it completely dissolvedwhen added at 400 g/L and 440 g/L.

The results from the SDS PAGE indicated that the different levels ofammonium sulphate used for the precipitation step (400 g/L, 440 g/L, 480g/L, 520 g/L) or the other methods used (oxygenation, pH shift) did notseem to have an obvious effect on the amounts of collagenases present inthe post dialyzed samples. In all cases, the concentration of each ofthe collagenases in the post dialyzed samples ranged between 50 mg/L and60 mg/L. FIG. 18a shows a representative SDS PAGE gel, such as that ofthe post dialyzed sample precipitated with 400 g/L ammonium sulphate.Since all the gels were very similar the other SDS PAGE gels are notpresented in this report.

Taking into account the estimated concentrations of collagenases in theharvest point sample (FIG. 17) and in the post dialyzed samples, therecovery of the collagenase after the precipitation/dialysis steps wasapproximately 50%. In order to investigate whether the value of 50%recovery was accurate, since the error in the estimation of collagenaseconcentration by SDS gel is in general high, the following SDS PAGE gelswere carried out.

-   -   An SDS PAGE gel of all the supernatants after centrifugation of        the ammonium sulphate precipitated samples (FIG. 18a ). The aim        was to assess whether any amount of collagenases is lost into        the supernatant.    -   An SDS PAGE gel in which the harvest point supernatant sample        and the post dialysed ammonium sulphate (400 g/L) precipitated        sample were appropriately diluted to contain equal amounts of        collagenases and loaded on the same gel (FIG. 19).    -   An SDS PAGE gel in which the harvest point supernatant sample        and the post dialysed ammonium sulphate sample (520 g/L) were        appropriately diluted to contain equal amounts of collagenases        and loaded on the same gel (FIG. 20).        It can be seen from FIG. 18b that the amount of collagenases        present in the supernatants after centrifugation of the ammonium        sulphate precipitated samples was very low. In FIG. 19 and FIG.        20 that the amount of collagenases after the        precipitation/dialysis steps appeared to be very similar to that        in the supernatant harvest sample. It was therefore likely that        the recovery value that was derived by comparing the        semi-quantitative SDS PAGE gels of the supernatant and the        post-dialyzed samples was actually higher.        Benchmarking Fermentation Experiments with Animal Derived        TSB/Proteose:

Fermentations of C. histolyticum 013 and 004 strains in the mediacontaining animal derived components were performed. The aim was tocompare strain 013 to strain 004 and evaluate the effect of the animalcomponents on cell growth, collagenase expression and on the levels ofcontaminants.

C. histolyticum 013:

The lyophilised strain was re-constituted in PBS and plated out ontoTSB/Proteose agar plates (30 g/L TSB, 10 g/L proteose peptone, 12 g/Lagar. The plates were incubated in an anaerobic jar in the presence ofanaerobic gas packs. Single colonies were picked and used to inoculate 5mL TSB/Proteose media. After 15 hours of incubation at 37° C. theOD_(600 nm) of the culture was approximately 1.0 unit. 5 mL of culturewas then mixed with 1 mL of sterile and stored below −70° C.

PBFT58 Fermentations Growth Curves:

Two 5 L batch fermentations were carried out, PBFT58c (strain 004) andPBFT58d (strain 013). Table 6 presents the recipe of the TSB/Proteosemedia used. FIG. 21 shows the growth curves obtained (Net OD_(600 nm) vstime).

TABLE 6 Recipe for TSB/Proteose media Component Concentration Proteosepeptone 50 g/L TSB 15 g/L MgSO₄•7H₂O 0.08 g/L KH₂PO₄ 1.92 g/L Na₂HPO₄3.5 g/L Vitamin solution 10 mL/L (Sterile filtered)

It was seen from FIG. 21 that the strain 013 grew to a higher OD thanstrain 004. In both cases however the final OD_(600 nm) was higher than2.5, indicating that the animal derived media supported good growth forboth strains.

It was noted that strain 013 continued to grow slowly up to the harvestpoint (20 hours) whereas strain 004 grew up to a net OD_(600 nm) ofapproximately 2.7 and then stopped growing. Compared to the fed-batchfermentations presented previously, using the non-animal derived media,the final OD obtained using the animal derived TSB/Proteose media waslower.

SDS PAGE Analysis:

The SDS PAGE gels (8% Tris-Glycine gels) of the supernatant samplestaken throughout the fermentations are shown in FIG. 22 and FIG. 23.

There did not seem to be any clostripain in the fermentationsupernatants, especially in the case of strain 013. This was a veryimportant finding since it could explain the fact that the originatormay not have had issues or reduced issues during the purification ofcollagenases. In contrast, significant problems with degradation of thecollagenases had been previously experienced during the purificationprocess. This could be partly attributed to the presence of clostripainin the fermentation.

In order to obtain a better estimate of the amount of collagenasespresent in the fermentations, a semi-quantitative SDS PAGE gel wasconduced for the harvest point samples (FIG. 24 and FIG. 25). The gelssuggest that lower amount of collagenases was produced in the batchfermentations with the TSB/Proteose media (PBFT58c) compared to thefed-batch fermentation with the vegetable media (PBFT57). This could beattributed to the fact that higher cell densities were obtained in thelatter case (OD_(600 nm)˜4 to OD_(600 nm)˜2.7). Table 7 summarizes theresults from the semi-quantitative gels.

TABLE 7 Results from semi-quantitative SDS PAGE gels for PBFT57 andPBFT58c, d PBFT57 PBFT58c PBFT58d (Animal-free, (Animal-derived,(Animal-derived, strain 004) strain 004) strain 013) AUX I (mg/L) 132 8859 AUX II (mg/L) 142 95 95 Total 274 183 154

Ammonium Sulphate Precipitation of Fermentation Harvest Samples:

For each fermentation, 2×500 mL harvest point samples were precipitatedwith 400 g/L (60%) and 520 g/L (80%) ammonium sulphate. The pellets werere-suspended in 16.5 mL of WFI and dialyzed against 100 mM of K₂HPO₄ (pH6.7). SDS PAGE analysis (8% Tris-Glycine gels) of the post-dialyzedsamples was then performed (FIG. 26 and FIG. 27).

The results from these gels indicated that the levels of clostripain,even in the very concentrated post-dialyzed samples (lanes 6 and 7 ofFIGS. 26 and 27) were extremely low. This is more evident in the case ofstrain 013 compared to strain 004.

Thus the invention relates to collagenase compositions which are free ofclostripain, such as those produced by the fermentation processesdescribed herein.

Measurement of Clostripain Activity:

In order to investigate further the role of clostripain an enzymaticassay was set up to measure the clostripain activity of post dialyzedsamples. The following method was used: Enzymatic assay of clostripain:

BAEE=N-a-Benzoyl-L-Arginine Ethyl Ester

Conditions: T=25° C., pH=7.6, A_(253 nm), Light path=1 cm

Method: Continuous Spectrophotometric Rate Determination

Unit definition: One unit will hydrolyze 1.0 μmole of BAEE per minute atpH 7.6 at 25° C. in the presence of 2.5 mM DTT.

Analysis of Post Dialysed Samples for Clostripain Activity:

The clostripain activity assay was used to analyze the post-dialyzedsamples from the fermentations with the TSB/Proteose (PBFT58) and thevegetable based fed-batch fermentation (PBFT57). Table 8 summarizes theresults.

The results demonstrate that there was very low clostripain activity inthe case of TSB/Proteose fermentations. In contrast the clostripainactivity in the case of the fed-batch PBFT58 was very high.

TABLE 8 Enzymatic activities of post-dialyzed samples PBFT57 PBFT58cPBFT58d (Animal-free, (Animal-derived, (Animal-derived, strain 004)strain 004) strain 013) Clostripain   56.4 *   1.0 *   0.1 * activity(U/mL) Specific 205.8 5.5 0.7 clostripain activity (U per mg totalcollagenase) * Clostripain activity determined in the postprecipitated/dialyzed sample

Investigation of Alternative Peptones Screening Experiments in ShakeFlask:

In this work various vegetable peptones were used as alternatives to thephytone peptone. The aim was to evaluate their effect on the levels ofexpression of the collagenases and clostripain. All the peptones testedare derived from vegetable sources and are marketed by Sigma.

The experimental procedure used is described in FIG. 28. The mediarecipes are detailed in Table 9, whereas a list of the peptones used isshown in Table 10. A control shake flask was also conducted, containingphytone peptone. In all cases, 50 g/L of yeast extract and 100 g/L ofeach peptone were used in an effort to mimic the concentrations of thesecomponents at the harvest point of the developed fed-batch fermentation(see Table 4).

TABLE 9 Composition of media used in shake flask experiment. All mediawere filter sterilised. Vegetable media Component ConcentrationAlternative Peptone 100 g/L Yeast extract 50 g/L KH₂PO₄ 1.92 g/L K₂HPO₄1.25 g/L Na₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/L Vitaminsolution 10 mL/L Glucose 0.9 g/L

The shake flasks were incubated for 18 hours. The cultures were analysedfor OD_(600 nm) and viable cell counts. The cultures were filtered andthe supernatants analysed by SDS PAGE. The results from the OD_(600 nm)measurements and viable cell counts are summarised in Table 10.

Most of the vegetable peptones resulted in higher net OD values comparedto the phytone peptone. However, the OD values did not correlate to theviable cell counts. This could be partly attributed to the variabilityof the viable cell count method or to the fact that the cells hadalready started to lyse before the pre-selected harvest point (18hours).

Interestingly, the SDS-PAGE gel indicated that there was no expressionof collagenase (gel not shown) in all the flasks, including that of thecontrol (phytone peptone). A possible reason for this could be the factthat the concentrations of the phytone peptone and yeast extract usedwere very high and as a result they repressed the expression ofcollagenases.

TABLE 10 Results from 1^(st) screening experiment Net OD_(600 nm) Typeof peptone after 18 h growth CFU/mL Phytone peptone (control) 2.65 1.2 ×10⁹ Proteose peptone (vegetable) 2.58 7.4 × 10⁸ Tryptone (vegetable)3.05 4.8 × 10⁸ Vegetable extract 3.22 1.0 × 10⁹ Vegetable extract 1 3.119.6 × 10⁹ Vegetable extract 2 3.05 7.8 × 10⁹ Vegetable hydrolysate 23.01 8.4 × 10⁹

Fed-Batch Fermentations Using Alternative Peptones—DCFT27a,b:

Based on information from the previous shake flasks experiments that noexpression of collagenases was observed, it was decided to evaluate thealternative peptones using the developed fed-batch strategy.

Two fed-batch fermentations were conducted, DCFT27a (vegetable extract2) and DCFT27b (vegetable hydrolyzate 2). In both fermentations thefed-batch strategy that was developed for the media containing phytonepeptone was used. Table 11 describes the media recipes, whereas FIG. 29the strategy used.

TABLE 11 Media recipe for fed-batch fermentations DCFT27a and DCFT27bDCFT27a,b Concentrations at Component Batch phase Feed harvest pointAlternative Peptone 60 g/L 151.4 g/L 100 g/L Yeast extract 20 g/L 127.1g/L 50 g/L Glucose 7.5 g/L 17.8 g/L 10 g/L Filtered sterilized KH₂PO₄1.92 g/L K₂HPO₄ 1.25 g/L Na₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/LVitamin solution 10 mL/L Volume 3.6 L 1.4 L ~5 L

Growth Curves:

The growth curves (Net OD_(600 nm) vs. time) for DCFT27a and DCFT27b aredepicted in FIG. 30. In both fermentations, the cells grew to a slightlyhigher OD_(600 nm) compared to the media containing phytone peptone(fermentation PBFT57, FIG. 16). This was in accordance with the viablecell counts (approximately 2×10⁹ CFU/mL for DCFT27a,b compared to1.5×10⁹ CFU/mL for PBFT57).

SDS PAGE Gels:

As with the shake flask experiments the SDS PAGE analysis indicated thatthere was no expression of collagenases in both DCFT27a and DCFT27b(gels not shown). This could be attributed to the fact that the media,which consists of high amounts of peptone, supports the expression ofcollagenases when phytone peptone is used, but is too rich when analternative peptone is used and thus represses the expression of anymetabolite, including collagenase and clostripain. It seems that thecells experience luxurious growth conditions in the media containing thealternative peptones and do not need to produce any proteases.

Batch Fermentations Using Alternative Peptones—PBFT59a,b,c:

The results from DCFT27a and DCFT27b fed-batch fermentations, led tofurther work to investigate three additional alternative peptones,however using lower concentrations than previously used.

Three 5 L batch fermentations were conducted, PBFT59a (vegetabletryptone), PBFT59b (vegetable extract) and PBFT59c (vegetable extractno. 1). The fermentations were harvested after 18 hours.

All peptones were used at concentrations of 50 g/L in an effort to mimicthe concentration of the proteose peptone in the animal media(Proteose/Peptone) and the concentration of phytone peptone that wasused previously. The media recipe is shown in Table 12.

TABLE 12 Media recipe and fermentation strategy for 5 L fermentationsPBFT59a,b,c Component Concentration Alternative Peptone 50 g/L Yeastextract 8.5 g/L Glucose 5 g/L KH₂PO₄ 1.92 g/L K₂HPO₄ 1.25 g/L Na₂HPO₄3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/L Vitamin solution 10 mL/L Volume5 L

Growth Curves:

The growth curves obtained from PBFT59a,b,c fermentations are depictedin FIG. 31. In all cases the cells grew to a lower OD_(600 nm) (between1.8 and 2.8) compared to the DCFT27 fed-batch fermentations. This wasalso in accordance with the viable cell counts (between 0.7×10⁹ CFU/mLto 1.2×10⁹ CFU/mL for PBFT59a,b,c compared to 2×10⁹ CFU/mL forDCFT27a,b). In the media containing tryptone the cells demonstrated theslowest growth rate and achieved the lowest cell density after 18 hours.

SDS PAGE Gels:

As for the shake flask experiment and the DCFT27a,b fed-batchfermentations no collagenase expression was seen in the SDS PAGE gels(gels not shown).

These results show that the alternative peptones, although they supportthe cell growth, they do not allow the expression of collagenases. Assuggested before this could be due to the fact these peptones are veryrich in nutrients, e.g., free amino acids, small peptides.

4^(th) Set of Fed-Batch Fermentations—DCFT27d

As the results from the experiments using the alternative vegetablepeptones were not successful the next aim of this work was toinvestigate the possibility of decreasing the levels of clostripain inthe developed fed-batch fermentation using the phytone peptone media. Asdescribed previously, the clostripain was probably causing thedegradation of collagenases during the purification process.

A fed-batch fermentation was carried out using the standard phytonepeptone media supplemented with three amino acids, i.e., glutamine,tryptophan and asparagine. This fermentation was performed as theconcentrations of these particular amino acids were lower in the phytonepeptone compared to the animal TSB/Proteose media, based on the aminoacid composition of these components, provided by the manufacturers.

The aim here was to investigate whether addition of these amino acidscould reduce any nutrient limitation that may be a contributing factorfor the expression of clostripain. The media recipe is shown in Table13. The fermentation strategy used was the standard fed-batch strategyused for DCFT26 and PBFT57 fermentations (see FIG. 6).

TABLE 13 Media recipe for fed-batch fermentation DCFT27d DCFT27d BatchConcentrations at Component phase Feed harvest point Phytone Peptone 80g/L 151.4 g/L 100 g/L Yeast extract 20 g/L 127.1 g/L 50 g/L Glucose 7.5g/L 17.8 g/L 10 g/L Amino acids Glutamine 2.8 g/L Filtered Tryptophan0.35 g/L sterilised Asparginine 0.18 g/L KH₂PO₄ 1.92 g/L K₂HPO₄ 1.25 g/LNa₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/L Vitamin solution 10 mL/LVolume 3.6 L 1.4 L ~5 L

Growth Curve:

The growth curve obtained from DCFT27d fermentation is depicted in FIG.32. The growth profile obtained was very similar to that obtained forthe standard fed-batch fermentation in the absence of amino acids(DCFT26b and PBFT57) shown previously.

SDS PAGE Gel.

FIG. 33a shows the SDS PAGE gel of the supernatant samples takenthroughout the fermentation. The level of collagenases is similar tothat seen for the standard fed-batch fermentation (see FIG. 10 for SDSPAGE gel from DCFT26b). Although clostripain is still present in thefermentation, it did seem that its level was lower than that in DCFT26b.

In order to investigate this further, the clostripain activity of thepost-dialysed harvest point sample was estimated using the clostripainactivity assay. In addition, the clostripain activity of thepost-dialysed harvest point sample taken from the 20 L lyophilizationbatch was also estimated. Since this particular batch was purifiedwithout showing significant collagenase degradation, knowledge of itsclostripain activity would be informative. Table 14 summarizes theenzymatic activities of the post-dialyzed samples. It also includes theenzymatic activities for the standard fed-batch fermentation PBFT57 andthe animal TSB/Proteose peptone presented in Table 8, for comparativepurposes.

TABLE 14 Enzymatic activities of post-dialyzed samples DCFT27d PBFT57Fed-batch PBFT58c Standard fed- plus amino 20 L Lyo Animal TSB/ batchacids batch Proteose Clostripain 56.4 15.2 16.6 1.0 activity (U/mL)Specific 205.8 55.3 184.4 5.5 clostripain activity (U per mg totalcollagenase)

The results from DCFT27d indicate that the addition of the amino acidsreduces the activity of clostripain produced by the strain. The ratio ofclostripain to collagenase is approximately four fold lower in the aminoacid supplemented fermentation compared to the control fed-batchfermentation. The ratio of clostripain to collagenase in theanimal-derived fermentation was ten fold lower than the amino acidsupplemented fed-batch fermentation. It is possible that the reductionof clostripain activity may result in significant reduction on thedegradation of collagenases during purification.

A series of 5 L fermentations were conducted to assess several fed-batchfermentation strategies. The strategies were assessed based on theiryield of collagenase, quantity of contaminants and scalability. Based onthese results an optimum fed-batch strategy was identified that resultedin a productivity of total collagenases of approximately 280 mg/L. Thefermentation strategy was modified by slightly increasing the batchmedia concentration and reducing the fed-batch media concentration toimprove its scalability. This change to the fermentation strategy had noeffect on the productivity or levels of contaminants.

The second objective was to optimize the primary recovery step of thecollagenases. Optimization of this step involved improvement in theyield of the process step or a reduction in the quantity of contaminantsrecovered or an increase in scalability. A range of ammonium sulphateconcentrations from 100 to 520 g/L were assessed. The effect of loweringthe pH to 6.0 and oxygenating the media were also assessed. All ammoniumsulphate concentrations below 400 g/L showed very low recoveries ofcollagenase. No difference in the recovery of collagenase or clostripainwas observed in any of the ammonium sulphate concentrations between 400and 520 g/L. The pellet from the 400 g/L precipitation was the easiestto re-suspend and this concentration was therefore defined as theoptimum level.

A benchmarking experiment was carried out in order to determine andcompare the growth and production of collagenases and clostripain in ananimal-derived media with C. histolyticum strains 013 and 004. Theanimal-derived media recipe was taken from the Process 1 fermentationmedia, utilizing TSB and protease peptone. This experiment also alloweda comparison of strain 004 grown in animal and non-animal media. Theresults from SDS-PAGE analysis showed that much lower quantities ofclostripain from C. histolyticum grown in the animal-derived media.These results were confirmed using an enzymatic assay for clostripainactivity. The assay demonstrated a significant reduction in the activityof clostripain in fermentations using the animal-derived media. When thetwo strains were compared 004 showed a higher clostripain activity than013.

Selections of alternative nitrogen sources were assessed for theirability to replace the Phytone peptone in the fed-batch fermentationstrategy. These peptones were Vegetable Extract No. 2 (Sigma, 49869) andVegetable Hydrolysate No. 2 (Sigma, 07436). The C. histolyticum grewextremely well on the vegetable peptones reaching optical densities (600nm) of 4 to 5 units. SDS-PAGE analysis of these fermentations showed noexpression of either collagenase or clostripain. Due to the luxuriantcell growth observed on these peptones it was thought that theconcentration of complex nitrogen source was too high resulting in aninhibition of protease expression. A second set of fermentations wastherefore carried out using the alternative peptones at 50 g/L in abatch strategy. Vegetable Tryptone (Sigma, 16922) Vegetable Extract(Sigma, 05138) and Vegetable Extract No. 1 (Sigma, 04316) were used asalternative peptones for these experiments. When the fermentations wereanalyzed by SDS-PAGE no expression of collagenase or clostripain wasseen. A fed-batch fermentation using Phytone peptone was supplementedwith three amino acids, glutamine, tryptophan and asparagine. Theseamino acids were identified as being present in lower amounts in thenon-animal media. The growth profile of the fermentation was verysimilar to that of the fed-batch fermentation without amino acidsupplementation. SDS-PAGE analysis showed a similar yield of collagenasebut a slightly lower level of clostripain. The clostripain assay showedreduced activity in the amino supplemented when compared to the controlfed-batch fermentation. The reduction in clostripain activity whilststill significant was not as great as the difference between animal andnon-animal media.

Materials and Methods: Inoculum Media for Fermentations Using VegetableMedia

Throughout this development work the following recipes for the inoculummedia were used.

Inoculum media - Vegetable Component Concentration Vegetable Peptone 50g/L Yeast extract 8.5 g/L Glucose 0.9 g/L KH₂PO₄ 1.92 g/L K₂HPO₄ 1.25g/L Na₂HPO₄ 3.5 g/L NaCl 2.5 g/L Magnesium 0.08 g/L Vitamin solution 10mL/LThe media was filter sterilized

Inoculation Procedure

A vial from the internal cell bank was thawed and 0.025 mL was used toinoculate 5 mL of the inoculum media in a 30 mL universal. The 5 mLculture was incubated at 37° C. in an anaerobic jar in the presence ofanaerobic gas generators. After approximately 13 to 15 hours ofincubation, 4 mL of the culture was used to inoculate 200 mL of theinoculum media in a 500 mL flask. As previously the flask was placed inan anaerobic jar in the presence of anaerobic gas generators. Afterapproximately 13 to 15 hours of incubation at 37° C. and 75 rpm, thewhole content of the flask was used to inoculate the fermenter.

The pH and the temperature of the fermenters were controlled at 7.0 and37° C., respectively. The nitrogen flow rate was set at 1 L/min (˜0.2vvm) and the stirrer speed at 100 rpm. The fermenter was sampled atregular time intervals for OD_(600 nm) measurements and viable cellcounts. Samples were filtered through a 0.22 μm filter. The filtrateswere stored at −20° C. and were frozen at −20° C. for SDS PAGE analysis.FIG. 33b depicts a schematic diagram the inoculation procedure.

A preferred recipe for the fed-batch fermentation is set forth below.

Component Quantity Batch Phase KH₂PO₄ 2.91 g/L K₂HPO₄ 1.89 g/L Na₂HPO₄5.30 g/L NaCl 3.79 g/L Phytone 65.45 g/L Bacto Yeast Extract 21.80 g/LMgSO₄ 0.12 g/L FeSO₄ × 7H₂O 18.18 mg/L Riboflavin 0.758 mg/L Niacin 1.52mg/L Calcium Pantothenate 1.52 mg/L Pimelic acid 1.52 mg/L Pyridoxine1.52 mg/L Thiamine 1.52 mg/L Volume for 5 L fermentation 3.3 L Fed-batchPhase Glucose 17.86 g/L Phytone 151.43 g/L Bacto Yeast Extract 127.14g/L Volume for 5 L fermentation 1.4 L

It is also desirable to scale-up the fermentation process furtherwithout detracting from the quality or yields of the collagenaseproducts. Thus, the invention further relates to an approximately 200liter fed batch process as described in the flow chart in FIG. 33 c.

Viable Cell Counting Method

Samples taken from the shake flasks were diluted by a factor of 10⁻⁴ to10⁻⁷ and plated out onto TB agar plates. Plates were incubated at 37° C.for approximately 48 hours in a Genbox Jar. An Anaerobic Gas GeneratorPack was used in order to create anaerobic conditions within the Jar.The number of colonies was then counted.

Ammonium Sulphate Precipitation:

Materials: Sorvall Evolution centrifugeChemicals: Ammonium Sulphate, GPR grade (BDH)

Supernatant samples (100 mL to 500 mL) were filtered through a 0.22 μmfilter. Depending on the experiment various amounts of ammonium sulphatewere added (from 15% to 80% saturation). The solution was mixed slowlyin a magnetic stirrer for approximately 15 minutes, until all theammonium sulphate had dissolved. It was then held without mixing for˜3.5 hours at +2-8° C. Following the hold step, significant amount ofprecipitate was formed. The solution was then centrifuged at 7,200×g for20 minutes at 4° C. The supernatant was decanted and the pellet storedat −20° C.

Dialysis Materials: 10 kDa MWCO SnakeSkin Dialysis Tubing (68100,Pierce) Magnetic Stirrer Chemicals: Potassium Dihydrogen OrthophosphateAnalaR (BDH) Water for Injection (WFI)

The pellets obtained from a 100 mL ammonium sulphate sample werere-suspended in 3.3 mL of WFI. The re-constituted pellet was transferredinto a pre-wetted 10 kDa MWCO SnakeSkin dialysis tubing and dialyzedagainst 100 mM of K₂HPO₄ (pH 6.7) for ˜12 to 16 hours at 2-8° C. The WFIwas then changed and dialysis continued for 2 to 4 hours. The dialyzedmaterial was recovered and the volume determined. The post-dialyzedsample was stored at −20° C.

SDS-PAGE Analysis (8% Tris-Glycine Gels) Materials: Xcell SureLockMini-Cell Chemicals: SDS-PAGE Standards High Molecular Weight (161-0303,Bio Rad)

Novex 8% Tris-Glycine gels, 1.5 mm, 10 well (EC6018BOX, Invitrogen)Novex 8% Tris-Glycine gels, 1.5 mm, 15 well (EC60185BOX, Invitrogen)

Novex Tris-Glycine SDS Running Buffer (10×) (LC2675, Invitrogen) NovexTris-Glycine SDS Sample Buffer (2×) (LC2676, Invitrogen) NuPAGE SampleReducing Agent (10×) (NP0009, Invitrogen) Collodial Blue Staining kit(LC6025, Invitrogen)

Ethylenediaminetetra-acetic acid disodium salt Analar R (BDH)

Samples were prepared for reducing SDS-PAGE by adding 10 μl of sample to10 μl sample Buffer (2×), 2.5 μl reducing agent (10×) and 2 μl of 0.1MEDTA (to achieve final concentration of 10 mM). The high molecularweight (HMW) marker was prepared by adding 10 μl of concentrated stockto 80 μl reducing agent (10×), 310 μl WFI and 400 μl sample buffer (2×).The diluted HMW standard was then heated at 95° C. for 5 minutes beforealiquoting and storage at −20° C. for use in subsequent gels. Samples(15 μl) containing collagenases were run directly (i.e. with no priorheat treatment) on 8% Tris-Glycine gels using Tris-Glycine runningbuffer at 130V for ˜1 hour 50 mins. After electrophoresis, the gels werestained with colloidal blue stain reagent as per the manufacturer'sinstructions.

Purification Process Method Summary for 5 L Process of Purification:

Step 1. Ammonium sulfate precipitation of culture media supernatant(secreted protein) Reconstitution and dialysis into 0.1M potassiumphosphate, 0.1M arginine pH 6.7 Step 2. Hydroxyapatite chromatography(in presence of 200 μM leupeptin) Elute with 0-100% gradient of 0.264 Mpotassium phosphate pH 6.7 over 4 CV Pool 2 late-eluting peaks whereA₂₈₀ > A₂₆₀, load straight onto TMAE Step 3. Fractogel TMAE ion exchange(in presence of 200 μM leupeptin) Nucleic acid removal (a Pall MUSTANG Qfilter can also be used) Collect and pool unbound flowthrough Step 4.Dialysis into 10 mM Tris pH 8.0 Step 5. Q Sepharose HP ion exchange (inpresence of 200 μM leupeptin) Separates AUXI from AUXII Elute with 0-40%gradient of 10 mM Tris, 3 mM CaCl₂, 360 mM NaCl pH 8.0 over 20 CV 2peaks collected:  Peak 1 = AUXII          Peak 2 = AUXI Arginine addedto 0.1M to AUXI and AUXII containing fractions Step 6. AUXI and AUXIIpools concentrated by pressurized stirred-cell Step 7. Superdex 75 GelFiltration Removal of clostripain and gelatinase from AUXI and AUXIIAUXI and AUXII run individually on separate columns Samples loaded at 5%CV Buffer: 10 mM Tris, 3 mM CaCl₂, 150 mM NaCl, 0.1M Arginine pH 8 Step8. The AUXI and AUXII are pooled and concentrated individually,diafiltered into water and then pooled to form the final drug product.

Column Details:

TABLE 15 Column specifications for 5 L process Volume Bed height Media(mL) Column (cm) Asymmetry Plates/meter HA 300 XK50/30 15 1.85 9227Fractogel 58 XK26/40 10 1.02 5368 TMAE Q 100 XK50/20 5 1.35 19,367Sepharose Superdex 880 XK50/60 45 1.24 18,644 75-1 Superdex 880 XK50/6045 1.85 13,576 75-2

Column Packing

-   -   Columns were packed as manufacturer's instructions where        possible.    -   TMAE column—no issues were encountered.    -   Q Sepharose and Superdex 75—difficulties were encountered in        packing to correct pressure due to size of the column. However,        the columns could be run at the recommended pressure.    -   HA—packed as a 50% slurry and run at 10 mL/min.        Yields/Recoveries from 5 L Process:

TABLE 16 Purification from AS ppt to Q-Sepharose IEX Chromatography stepyields in bold Protein Total Concentration Volume Protein Step ProcessStep (mg/mL) Method (g) (mg) Yield (%) Post AS ppt 1.12 Bradford 346.45388.02 — and dialysis Pre HA 1.08 Bradford 359.85 388.64 — (post-leupeptin addition) Post HA 0.51 Bradford 646.85 329.89 84.88 Pre-TMAE0.51 Bradford 646.85 329.89 — Post-TMAE 0.51 UV 647.2 330.07 100.05 Postdialysis 0.404 UV 715.0 288.86 87.51 Pre-IEX 0.388 UV 744.0 288.67 —Post IEX 0.454 UV 188.1 85.40 29.58 ABC I (peak 2) Post IEX 0.536 UV220.7 118.29 40.98 ABC II (peak 1)

TABLE 17 Purification from Q-Sepharose IEX to post Superdex 75 GPCProtein Total Concentration Volume Protein Step Process Step (mg/mL)Method (g) (mg) Yield (%) Pre-stirred 0.454 UV 188.1 85.40 — cell ABC IPost-stirred 1.901 UV 41.6 79.08 92.6 cell ABC I Pre GPC 1.901 UV 40.677.18 — ABC I Post GPC 1.12 UV 60.0 67.2 87.07 ABC I Pre-stirred 0.536UV 220.7 118.29 — cell ABC II Post-stirred 2.76 UV 45.5 125.58 106.16cell ABC II Pre GPC 2.46 UV 44.0 108.24 — ABC II Post GPC 1.192 UV 59.370.68 65.3 ABC IIYields from a 5 L process are approximately 60-75 mg each of ABCI andABCII For the scale up, depending on fermentation, yields of 250-300 mgfor 20 L and 2500-3000 mg for 200 L could be expected.

Individual Chromatography Steps of 5 L Scale Process: HydroxyapatiteChromatography

-   Column size: 2×300 mL in XK50/30 (15 cm bed height each)-   Buffer A: 0.1M potassium phosphate, 200 μM leupeptin, pH6.7-   Buffer B: 0.264M potassium phosphate, 200 μM leupeptin, pH6.7-   Sample: ˜350 mL (in 0.1M potassium phosphate, 0.1M Arginine pH6.7)    loaded at <1.0 mg/mL media*-   Flow rate: 9.8 mL/min-   Elution: 0-100% B over 4 CV

FIG. 34 shows a chromatogram after hydroxyapatite with a loading of 1.0mg/L media, wherein a considerable loss of resolution and targetdegradation occurs.

Fractogel TMAE Anion Exchange

-   Column size: 58 mL in XK26/20 (10 cm bed height)-   Buffer A: 10 mM Potassium Phosphate, 0.2M NaCl, 200 μM leupeptin,    pH6.7-   Buffer B: 10 mM Potassium Phosphate, 2M NaCl, pH6.7-   Sample: ˜650 mL @ 0.5 mg/mL (in Potassium Phosphate pH6.7, straight    from HA column) loaded at −5.5 mg/mL media-   Flow rate: 8.8 mL/min-   Elution: (100% B to elute nucleic acid)

FIG. 35 illustrates a chromatogram after Fractogel TMAE anion exchange.The unbound fraction pooled to give ˜650 mL at 0.5 mg/mL. Dialysed into10 mM Tris at pH8.

FIG. 36 shows a SDS-PAGE gel of Pre HA, Post HA and Post TMAE materialfrom 5 L scale process. The gel is stained with Colloidal blue.

Q Sepharose HP Anion Exchange with Original Elution Gradient

-   Column size: 100 mL in XK50/20 (5.0 cm bed height)-   Buffer A: 10 mM Tris, 3 mM CaCl₂, 200 μM leupeptin, pH8.0-   Buffer B: 10 mM Tris, 3 mM CaCl₂, 360 mM NaCl, 200 μM leupeptin,    pH8.0-   Sample: ˜650 mL at 0.5 mg/mL (in 10 mM Tris, pH8.0+200 μM leupeptin)    loaded at ˜3.0 mg/mL media-   Flow rate: 18.0 mL/min-   Elution: 0-40% B over 20 CV

FIG. 37 illustrates a chromatogram after Q Sepharose HP anion exchangewith original elution gradient. Arginine is added to 0.1M to ABCI andABCII containing fractions. Peak 1 fraction (ABCII) pooled to give ˜220mL at 0.55 mg/mL which was concentrated by stirred-cell to give ˜45 mLat 2.8 mg/mL. Peak 2 fractions (ABCI, excluding gelatinase shoulder)pooled to give ˜190 mL at 0.45 mg/mL, which was concentrated bystirred-cell to give ˜42 mL at 2 mg/mL.

FIG. 38 shows a SDS-PAGE gel of Q Sepharose IEX chromatography of postTMAE material run in the presence of leupeptin for Peak 1 (ABCII). Thegel is stained with Colloidal blue.

FIG. 39 shows a SDS-PAGE gel of Q Sepharose IEX chromatography of postTMAE material run in the presence of leupeptin for Peak 2 (ABCI). Thegel is stained with Colloidal blue.

Q Sepharose HP Anion Exchange with Modified GradientSmall scale test of NaCl addition to Buffer A and using a steeper/fastergradient. Sample was from a 1/3 5 L process, post TMAE, previouslyfrozen (−20° C.).

-   Column size: 1 mL-   Buffer A: 10 mM Tris, 30 mM NaCl, 3 mM CaCl₂, 200 μM leupeptin,    pH8.0-   Buffer B: 10 mM Tris, 3 mM CaCl₂, 360 mM NaCl, 200 μM leupeptin,    pH8.0-   Sample: 3 mg post TMAE, post dialysis into 10 mM Tris, 30 mM NaCl,    200 μM leupeptin, pH 8.0. Loaded at 3 mg/mL media-   Gradient: 0-25% B over 2CV, 25% B for 2CV, 25-40% B over 7.5CV

FIG. 40 illustrates a chromatogram after Q Sepharose HP anion exchangewith modified elution gradient. Good separation of ABCI and ABCII isobserved. The second part of the gradient can be made steeper to sharpenABCI peak. Improvement of the peak can also be made using 5 mL CV loadedat 3 and 10 mg/mL media.

Superdex 75 Gel Permeation Chromatography of ABCII (Peak 1 from IEX)

-   Column size: 880 mL in XK50/60 (54 cm bed height)-   Buffer: 10 mM Tris, 3 mM CaCl₂, 150 mM NaCl, 0.1M arginine, pH8.0-   Sample: ˜44 mL (5% CV) at 2.5 mg/mL (in 10 mM Tris, 3 mM CaCl₂, ˜60    mM NaCl, 0.1M arginine, pH8.0)-   Flow rate: 8.8 mL/min

FIG. 41 illustrates a chromatogram after superdex 75 gel permeationchromatography of ABCII (Peak 1 from IEX). Peak pooled to give ˜60 mLABC II at 1.2 mg/mL.

FIG. 42 shows a SDS-PAGE gel of superdex 75 gel permeationchromatography of concentrated ABC II run in the presence of arginine.The gel is stained with Colloidal blue.

Superdex 75 Gel Permeation Chromatography of ABCI (Peak 2 from IEX):

-   Column size: 880 mL in XK50/60 (54 cm bed height)-   Buffer: 10 mM Tris, 3 mM CaCl₂, 150 mM NaCl, 0.1M arginine, pH8.0-   Sample: ˜42 mL (5% CV) at 2.0 mg/mL (in 10 mM Tris, 3 mM CaCl₂, ˜60    mM NaCl, 0.1M arginine, pH8.0)-   Flow rate: 8.8 mL/min

FIG. 43 illustrates a chromatogram after superdex 75 gel permeationchromatography of ABCI (Peak 2 from IEX). Peak pooled to give ˜60 mL ABCI at 1.1 mg/mL.

FIG. 44 shows a SDS-PAGE gel of superdex 75 gel permeationchromatography of concentrated ABC I run in the presence of arginine.The gel is stained with Colloidal blue.

Scale Up Column Sizing

TABLE 18 Hydroxyapatite Fractogel TMAE Process Media Column Bed MediaColumn Bed Scale volume type height volume type height 1/3 220 mLXK50/30 ~11 cm 18 mL XK16/20  ~9 cm  5 L 2 × 300 mL XK50/30 ~15 cm 54 mLXK26/40 ~10 cm  20 L 2.4 L (at 1 * * 216 mL XK50/20 ~11 cm mg/mL load)200 L 24 L * * 2.2 L * * Q Sepharose HP Superdex 75 Process Media ColumnBed Media Column Bed Scale volume type height volume type height 1/3  65mL XK26/20 ~12 cm 300 mL XK26/70 ~57 cm  5 L 100 mL XK50/20  ~5 cm 880mL XK50/60 ~45 cm  20 L 400 mL (at 3 * * 4 L * * mg/mL load) 200 L 4 L(at 3 * * 40 L * * mg/mL load) * Column type and resulting bed height tobe further optimized. Media volumes are linear scale up from 5 L scale.

In yet other embodiments of the invention, the dialysis steps of thepurification process described above can be substituted withultrafiltration/diafiltration (UF/DF) operations using dialysis andstirred cells will be replaced by TFF, tangential flow filtration. TheTMAE step discussed above is optional.

The invention includes the collagenase products that are produced by (orcan be produced by) the above purification processes. Such collagenaseproducts possess exceptional high degrees of purity and retainedenzymatic activity. For example, the compositions are free ofclostripain (e.g., possess negligible or undetectable levels ofclostripain).

Optimization of the Manufacturing Process:

In order to support clinical studies and provide a commercial-scaleprocess, optimization of the manufacturing process earlier developed wascompleted. The process changes are described briefly below, and areoutlined in Table 19.

TABLE 19 Summary of Process Changes between BTC (Process 1) and AuxiliumSupplies (Process 2 and 3) Stage Process 1 Process 2 Process 3Fermentation Cell line 013 and 004 004 004 and Primary Cell line storageform Lyophilized Frozen liquid culture Frozen liquid culture RecoveryCell bank medium Bovine-derived Non animal-derived Non animal-derivedSeed medium Bovine-derived Non animal-derived Proteose peptone(porcine-derived) Seed scale-up strategy 1 WCB vial → 1 × 500 mL 2 WCBvials → 2 × 30 mL → 1 WCB vials → 3 × 25 mL bottle → 45 L 2 × 500 mLflasks → 1 × 20 L → 4 × 200 mL flasks → fermentor fermentor 1 × 20 Lfermentor → 200 L fermentor Production medium Bovine-derived Nonanimal-derived Proteose peptone (porcine-derived) Production mediumAutoclaved Sartoclear maxicap and 0.2 μm In situ media sterilizationsterilization filters and 0.2 micron filtration Production strategyBatch Fed-batch Batch Production scale 45 L 20 L 200 L Harvest method 10μm and 1 μm filter Millipore Millistak HC Pod Millipore Millistak HC Podtrain filter Ammonium Sulfate 95% saturation 60% saturation Captureproteins using precipitation (AS ppt) Phenyl Sepharose FF LowSubstitution chromatography media Resuspended AS ppt buffer DialysisDialysis N/A exchange Temperature control None 2-8° C. solutions 2-8° C.solutions Purification New chromatography/filtration NA Mustang Q filterMustang Q filter step New chromatography step NA Superdex 75 GPCElimination of Hydroxyapatite (HA) and GPC HA and Q HP buffer systemsminus leupeptin 200 μM leupeptin 200 μM leupeptin Temperature controlNone 2-8° C. buffers and column 2-8° C. buffers and packings columnpackings Buffer exchange Pre-Q HP Dialysis Dialysis TFF Buffer exchangePost-Q HP Dialysis Dialysis NA Concentrate/diafilter into final DialysisDialysis TFF formulation Scale-up all steps for 200 L NA NA 4.5 timesfermentation Formulation Formulation of Drug DS in WFI dilute DS in WFIdilute w/Lactose DS in 10 mM Tris, 60 mM Substance (DS) w/LactoseSucrose, pH 8.0

Fermentation Optimization

Removal of the bovine-derived raw materials from the original cell bankand fermentation process was carried out. Strain 004 of Clostridiumhistolyticum was propagated for use as the master cell bank based onpassage viability required for scale-up. The specifications andanalytical results for the master cell bank are captured in Table 20. Inorder to increase biomass and production of collagenase, a fed-batchfermentation strategy was developed utilizing animal-free raw materialsin the growth medium at a 20 Liter fermentation scale. Furtherfermentation scale-up to 200 Liter was observed to require the use of aporcine-derived media component (i.e., Proteose Peptone #3, infra) toassure consistent cell growth, collagenase expression, and an improvedimpurity profile. Subsequent changes were made to increase the yield andpurity of collagenase over the downstream process. These changes includethe addition of new separation and filtration strategies, as well asscale-up of the production equipment to support the 200 Liter batchfermentation scale. FIG. 45 depicts a flow chart of the fermentation forprocess 3.

TABLE 20 Analytical Specifications and Test Results for Master Cell BankTest Specification Result Identity Expected profile, >95% Expectedprofile, 99.9% ID ID Viable Count ≧1 × 10⁶ cfu/mL on TB 1.3 × 10⁷ cfu/mLon TB agar agar Purity Test No extraneous organisms No extraneousorganisms observed observed Colony Morphology (anaerobic Irregularshape, 1-2 mm Irregular, flat elevation, at 37° C.) in size, grey towhite undulate margin 1-2 mm color diameter (48 hr), grey, white colorGram Stain Gram positive rods Gram positive rods Phenol Red Dextrosefermentation Negative Negative Hydrogen Sulfide production PositivePositive Gelatinase Test Positive Positive Spore Test Negative Negativeon all media Growth in cooked meat media Positive Positive Growth inthioglycollate media Growth as finger-like Growth as finger-likeprojection projection Motility Test (MIO media) Non-motile Non-motileBacteriophage None detected No confirmed evidence of phage

Primary Recovery and Purification Optimization

Further development to optimize the primary recovery and downstreampurification process is being undertaken. Substitution of the ammoniumsulfate precipitation with phenyl sepharose fast flow low sub columnchromatography to capture the collagenases has been implemented toimprove yields, eliminate the use of bulk ammonium sulfate and toimprove aseptic processing.

With regards to purification, the Pall MUSTANG Q filter has beenimplemented for residual DNA and impurity clearance to further enhanceyields and simplify the production process train and validationrequirements. The Quaternary Amine Sepharose High Performance (Q HP)operating parameters have been optimized to eliminate the Gel PermeationChromatography (GPC) step. In addition to the process changes citedabove, the drug substance formulation has been modified to include 10 mMTris, 60 mM Sucrose, pH 8.0, improving both product solubility and drugsubstance and drug product stability.

The optimization process took place in two stages. The initial process(Process 2) utilizes an animal-free medium for all cell banking andfermentation stages with the fed-batch fermentation performed at the 20Liter scale. The downstream process has been adapted from Process 1 toinclude MUSTANG Q filtration for residual DNA removal and Superdex 75GPC for additional host cell contaminant clearance. Leupeptin has alsobeen added to the chromatography buffer systems to prevent proteolyticdegradation. Process 2 material has been bridged analytically withProcess 1 material (Table 21A), and was tested in a side-by-sidepre-clinical study outlined herein.

Process 2 material has been proposed for use in the early stage of thePhase 3 clinical program. The specifications for Process 2 intermediatesand drug substance are detailed in Tables 22 and 23 respectively.Further process, formulation and lyophilization development provided anoptimized manufacturing process (Process 3). These changes include theaddition of new separation and filtration strategies, as well asscale-up of the production equipment to support the 200 Liter batchfermentation scale as outlined in Table 19. FIG. 46 depicts a flow chartof the purification for process 3.

Declaration of Dose:

The initial in vitro potency assay was a bovine collagenase assay anddid not differentiate collagenase types I and II. This assay wasutilized for the material used in the open label, DUPY101 and DUPY 202clinical studies only, with the 0.58 mg dose typically resulting in apotency of 10,000 Units. Analysis of Process 1 material utilizing thecurrent separate in vitro potency assays for type I collagenase and typeII collagenase typically results in 1,700 to 3,500 Units/dose (0.58 mgdose) for type I collagenase and 43,000 to 69,000 Units/dose (0.58 mgdose) for type II collagenase. Analysis of Process 2 material utilizingthe current in vitro potency assays has confirmed that similar relativepotency values compared to Process 1 material are typically achieved.

Demonstration of Analytical Comparability Between Process 1 and Process2:

In order to support the changes between Process 1 and Process 2,comparability data have been submitted in the form of release testingand analytical characterization. These data are presented in Table 21.

Comparison of the intermediates, described as AUX-I and AUX-II, and drugsubstance from the previous process (Process 1; Reference) with aprocess of the invention (Process 2). This analytical comparison showsthat material manufactured from Process 2 is comparable to that madewith Process 1 (Table 21). In particular, the identity, potency andpurity between these materials are comparable.

The purity level of Process 2 intermediates is shown in FIG. 47, areduced SDS-PAGE Coomasie stained gel. The gel shows a single band foreach intermediate with no other minor bands evident. AUX-I has anapparent MW of 115 kDa and compares with the reference (ABC I), whileAUX-II has an apparent MW of 110 kDa and compares with the reference(ABC II). FIG. 48 shows a reduced SDS-PAGE Coomasie stained geldepicting drug substance. As with the intermediates, drug substancemanufactured by Process 2 compares with the reference (Process 1). Asilver stained SDS-PAGE gel is depicted in FIG. 49 furthersubstantiating the high purity level of the Process 2 drug substance. Insummary, the release testing and analytical characterization for theintermediates (AUX-I and AUX-II) and drug substance manufactured usingProcess 2 clearly demonstrates comparability with Process 1 (Reference)materials. Additionally, further release testing was performed onProcess 2 material and is listed in Table 21B. In conclusion, the directanalytical comparison between Process 1 and Process 2 materials (Table21), and the further intermediate and release testing (Table 22)indicate that Process 2 material is suitable for use in the humanstudies. Tables 23 and 24 further list the analytical specificationsresulting from Process 2 manufacturing process.

TABLE 21 Analytical comparability between (Process 1) and Auxilium(Process 2) intermediates and drug substance. Intermediate IntermediateDrug Drug Substance Test AUX-I AUX-II Substance Specification Identityby SDS- Conforms to Conforms to Conforms Major Major PAGE reference (seereference (see to collagenase collagenase attached) attached) referenceband band (see between 100-115 kDa; between 107-110 kDa; attached) nominor no minor bands bands Rat Tail Tendon 2310 units/mg — 2866 units/mg1700-3500 units/mg Collagen Assay for Potency (AUX-I) Process 1Reference 2704 units/mg — 2018 units/mg * Potency for Class II — 179704units/mg 50955 units/mg 43000-69000 units/mg Collagenases (AUX-II)Process 1 Reference — 174045 units/mg 58491 units/mg * Analysis ofproteins 100% main 100% main 100% main ≧99% main peak; using the Agilent1100 peak; 0% peak; 0% peak; 0% ≦1% aggregates by area HPLC System(Purity aggregates aggregates aggregates and aggregation by sizeexclusion chromatography) Process 1 Reference 87% main 90% mainIntermediates * peak; 13% peak; 10% used** aggregates aggregatesAnalysis of proteins 99% AUX-I; 100% AUX-II 100% 2 major peaks (AUX I &using the Agilent 1100 1% AUX-II AUX-I and AUX II), combined ≧97% HPLCSystem AUX-II by area; Retention times of (Identity and purity by AUX-Iand AUX-II within reverse phase liquid 5% of reference chromatography)Process 1 Reference 89.4% ABC-I; 93% ABC-II; Intermediates * 5.4%ABC-II; 0.5% ABC-I; used** 5.2% other 6.5% other Analysis of proteins<1% <1% <1% <1% by area using the Agilent 1100 HPLC System (Gelatinaseby anion exchange chromatography) Process 1 Reference <1% <1% <1% *Peptide Mapping by Peak pattern N/A Peak Conforms to reference TrypticDigest and conforms to pattern Reverse Phase HPLC Reference conforms toReference N- & C-terminal Sequence Sequence Not Conforms to referencesequencing identical to identical to required Reference*** Reference *Process 1 preliminary specifications not included here **Drug Substancenot available for these tests, limited supplies on hand ***N-terminalsequencing completed for AUX-I (identical to reference), but furtherdevelopment required for AUX-II as N-terminus appears to be blocked.

TABLE 22 Analytical results for Process 2 intermediates and drugsubstance Intermediate Intermediate Test AUX-I AUX-II Drug Substance pHof Solution Not required Not required 6.8 Protein Concentration Notrequired 1.54 mg/mL Not required by Bradford Assay Total Protein by 1.36mg/mL 1.39 mg/mL 1.41 mg/mL Absorbance Spectrophotometry Residual HostProtein Not required Not required Band pattern similar to ReferenceResidual Host DNA Not required Not required 2.9 ng/mL* Endotoxin Notrequired Not required 8.7 EU/mg Residual Leupeptin Not required Notrequired <1 μg/mL *Result is at the LOQ of the previous residual DNAmethod

TABLE 23 Analytical Specifications for Process 2 AUX-I and AUX-IIIntermediates Specification Test AUX-I AUX-II Appearance Clear colorlessand free Clear colorless and free from particulate matter fromparticulate matter *Endotoxin <10 EU/mL <10 EU/mL Identity (and purity)by SDS- Major band between 110-115 kDa, Major band between 107-110 kDa,PAGE (Reduced conditions, and no minor and no minor Coomasie and silverstained) bands bands *Total Protein by Absorbance 1.0-1.5 mg/mL 1.0-1.5mg/mL Spectroscopy SRC assay (AUX-I) 1900-3300 units/mg Not applicableGPA assay (AUX-II) Not applicable 4300-6400 units/mg Analysis ofProteins using ≧99% main peak; ≦1% ≧99% main peak; ≦1% the Agilent 1100HPLC aggregates by area aggregates by area System (Aggregation by sizeexclusion chromatography) *Analysis of Proteins using the ≧97% by area≧97% by area Agilent 1100 HPLC System (Purity by reverse phase liquidchromatography) Analysis of Proteins using the <1% by area <1% by areaAgilent 1100 HPLC System (Residual gelatinase by anion exchangechromatography) Analysis of Proteins using the <1% by area <1% by areaAgilent 1100 HPLC System (Residual clostripain by reverse phase liquidchromatography) Identity by Peptide Mapping Conforms to referenceConforms to reference Bioburden <100 CFU/mL <100 CFU/mL *Tests requiredfor provisional release of intermediates for further manufacturing

TABLE 24 Analytical Specifications for Process 2 Drug SubstanceSpecification Test AUX-I AUX-II Appearance Clear colorless andessentially free from particulate matter Potentiometric Measure of 6.0to 7.0 pH of Solution Endotoxin <10 EU/mL Identity (and purity) by Majorcollagenase band Major collagenase band SDS-PAGE (Reduced between100-115 kDa; between 107-110 kDa; no conditions, Coomasie and no minorbands minor bands silver stained) *Total Protein by 1.1-1.5 mg/mLAbsorbance Spectroscopy *SRC assay (AUX-I) 1700-3500 units/mg NA *GPAassay (AUX-II) NA 43000-69000 units/mg Residual host cell protein <10ppm Residual host cell DNA <10 pg/dose Analysis of Proteins using the≧99% main peak; ≦1% aggregates by area Agilent 1100 HPLC System(Aggregation by size exclusion chromatography) *Analysis of Proteinsusing 2 major peaks (AUX I & AUX II), combined ≧97% by the Agilent 1100HPLC area; Retention times of AUX-I and AUX-II within 5% System(Identity and purity of AA4500 reference by reverse phase liquidchromatography) Analysis of Proteins using <1% by area the Agilent 1100HPLC System (Residual clostripain by reverse phase liquid chromatographyAnalysis of Proteins using <1% by area the Agilent 1100 HPLC System(Residual gelatinase by anion exchange chromatography) Residualleupeptin by <1% by area reverse phase chromatography *Bioburden <1CFU/mL *Tests required for provisional release of Drug Substance forfurther manufacturing

Detailed Experimental for Process 3: Process 3 Fermentation:

The fermentation process using Phytone peptone employed during Process 2had shown significant variability during both supplies for DSPdevelopment and GMP manufacture.

During previous work an animal derived Proteose Peptone had been shownto support the growth of C. histolyticum very well. The animal derivedProteose Peptone culture produced significantly less clostripain thanobserved during Process 2 and expressed AUXI and AUXII at a 1:1 ratio.As a result a regulatory acceptable animal derived peptone, ProteosePeptone #3 from Becton Dickinson (PP3), was evaluated in 5 L fermenters.Initial comparison to the existing Phytone based process (Process 2)showed that using the PP3 at 50 g/L generated a high biomassconcentration with a rapid exponential growth rate. The fermentationresulted in a higher product yield of >350 mg/L total collagenaseopposed to ˜230 mg/L from Process 2 (by semi quantitative SDS-PAGEanalysis). Further fermentations using PP3 demonstrated thatsignificantly less clostripain was produced using the animal derivedfermentation medium. The first three fermentations (using one batch ofPP3) demonstrated very consistent growth profiles. When the product wasanalysed by SDS-PAGE the yield and purity of collagenase was found to bevery reproducible between the three fermentations.

To supply DSP with material for process development severalfermentations were conducted using PP3. For this supply material threedifferent batches of PP3 were used. It was noted that when two of thesebatches were used the growth profiles of the cultivations were notconsistent with previous PP3 fermentations and demonstrated variabilityin the growth profile between fermentations. A small scale investigationshowed that batch to batch variability in the PP3 caused this variation.The small scale study also demonstrated that an increase in the PP3concentration to 100 g/L would prevent this variation.

Two 5 L fermentations were conducted with 100 g/L PP3 using two batchesof the peptone, one that resulted in the typical growth profile and onewhich did not (as demonstrated during the small scale experiment). Theexperiment showed that the increase in concentration ensured that thetwo fermentations with different batches of PP3 were reproducible. Thegrowth profiles were highly similar and the product was expressed at asimilar yield and purity.

The optimized fermentation process utilizing 100 g/L PP3 was finallyscaled to 200 L. The 200 L growth profile was very similar to that seenat 5 L scale. SDS-PAGE analysis of the fermentation filtrate showed ahigh yield from the 200 L fermentation, ˜320 mg/L total collagenase (byquantitative densitometry analysis). The purity of the collagenaseproduct (post fermentation) was similar at both 5 L and 200 L scale. 20L of the 200 L fermentation filtrate was processed by the DSP group torepresent a partial scale-up for the downstream process (infra).

The Proteose Peptone #3 fermentation process (Process 3) generatedcollagenase with a higher yield and with less clostripain than theexisting Phytone process. At 100 g/L PP3 was shown to yield C.histolyticum cultivations with reproducible growth curves despite usingvarious batches of PP3. Both the yield and purity of collagenase werealso shown to be reproducible when using various lots of PP3.

Evaluation of Proteose Peptone #3 as a Raw Material for Production ofCollagenase from Clostridium histolyticum.

Due to the variability observed in fermentations utilising Phytonepeptone as a complex nitrogen source the suitability of Proteose Peptone#3 (Becton Dickinson, 212230) (PP3) was evaluated in 5 L fermentations.A simple batch strategy with 50 g/L PP3 was used. The exact mediumcomposition can be found in the materials and methods section.

FIG. 51 compares the growth curve of the 50 g/L PP3 (a lowerconcentration than the Phytone concentration in Process 2) fermentationto the Phytone fed-batch fermentation. The PP3 cultivation demonstratesa very rapid specific growth rate during exponential growth beforeentering stationary phase approximately 8 hours after inoculation. ThePP3 fermentation reached a maximum optical density (600 nm) of 4.7units. The culture was left for a further 12 hours in stationary phaseto monitor product formation/degradation.

FIG. 52 shows SDS-PAGE semi-quantitative analysis of the concentrationof the collagenase products from the 20 hour point of the PP3cultivation. FIG. 53 shows the same analysis for the Phytone fed-batchprocess. It can be observed that the PP3 fermentation generates moreproduct than the Phytone based process (an increase from 230 mg/L to 360mg/L total collagenase, based on the semi-quantitative analysis in FIGS.52 and 53). The PP3 culture also expressed AUXI and AUXII at a 1:1ratio, whereas the Process 2 produced the two proteins at a 1:1.6 ratio.

Reproducibility of Proteose Peptone #3 Batch Fermentation.

The reproducibility of the PP3 batch process was further examined usinglot #5354796 of Proteose Peptone #3. All three runs illustrated in FIG.54 demonstrate consistent growth profiles with a maximal optical density(600 nm) of approximately 4.5 obtained after 8 hours.

Semi-quantitative SDS-PAGE analysis of the harvest points of thefermentation showed that yield of total collagenase to be ˜350-400 mg/L.

The harvest point of the fermentation was also evaluated during thisstudy. The fermentations were harvested at 8, 11 and 20 hours. FIGS. 55and 56 show SDS-PAGE analysis of the time course of PP3 fermentationGCFT05d (harvested at 11 hours). The gel depicted in FIG. 55 has beenstained with colloidal blue and the gel in FIG. 56 has been silverstained. A third higher molecular weight band can be observed above thetwo collagenase bands on the gels in FIGS. 55 and 56. It is thought thatthis band corresponds to an AUXI precursor protein reported in theliterature. The precursor band is present during the exponential growthphase. At the end of exponential growth the precursor band decreases inintensity and is not present after 11 hours (in GCFT05d). The main lowermolecular weight contaminants can be seen on the silver stained gel atapproximately 90, 60, 55, 45 and 40 kDa. It must be noted that thesecontaminants are present at a low level and are only clearly detected onthe silver stained gel. The optimal harvest point for the fermentationwas determined to be ˜11 hours at this stage of development. FIG. 57shows SDS-PAGE analysis of samples from the time course of a standardPhytone fed-batch fermentation. A 40 kDa contaminant can be observed onthe gel in FIG. 57. This 40 kDa contaminant band from the Phytonefed-batch process was identified as the protease clostripain. Bycomparing the gels in FIGS. 55 and 57 it is possible to determine thatthe quantity of clostripain produced using the PP3 fermentation processis significantly lower than the Phytone based fermentation.

Generation of Supply Material for Downstream Process Development

To support downstream process (DSP) development several fermentationswere conducted using 50 g/L PP3. During these fermentations twodifferent lots of PP3 were used (U.S. Pat. Nos. 5,332,398 and5,325,635). FIG. 58 depicts the growth curves of these fermentations(shown in diamond) compared to a fermentation (shown in square) usinglot #5354796 (GCFT05d). The fermentations with the new batches of PP3display highly varied growth profiles. Although the initial growth ratesof the cultures are all very similar, the point at which they enterstationary phase and therefore the maximum biomass concentrations differconsiderably. The optical densities (600 nm) in the inoculum culturesshowed very little variation (OD600 of 5 mL stage; 2.9-3.6 units, OD600of 200 mL stage; 4.5-5.9 units) and no reduction from previous inoculausing PP3 lot #5354796. The variation and reduced optical density (600nm) only manifested itself in the final (fermentation) stage of thecultivation. This suggests that reason for the variation was a nutrientlimitation in the PP3 and the quantity of the limiting nutrient variedbetween batches of PP3.

Although these fermentations were successfully used for DSP developmentand SDS-PAGE analysis showed that there was not a huge variation in thequantity of collagenase produced (350-400 mg/L total collagenase basedon semi-quantitative SDS-PAGE analysis, data not shown) it was decidedthat it was still critical to investigate the reason for the variation.The variation in the growth profile would make it very difficult topredict a harvest point of the fermentation. There were also concernsthat nutrient limitation may induce expression of other proteases asseen with the Phytone fed-batch process and specifically the protease,clostripain.

Investigation into the Variation Between Batches of Proteose Peptone #3.

Initial work with PP3 had demonstrated a highly robust process with ahigher product yield and lower levels of the protease clostripain. Whennew batches of PP3 were employed it was observed that the processrobustness decreased significantly with highly variable growth profiles.A shake flask experiment was conducted to directly compare the threebatches of PP3 used so far (lots 5354796, 5325635 and 5332398). Theexperiment replicated the two stage inoculum process from the 5 Lprocess but replaced the final fermentation phase with another 200 mLculture. Having this third stage was critical, as the variation was onlyobserved in the final fermentation stage of the process in previousexperiments. The optical densities (600 nm) of the cultures weremeasured at each transfer stage and the cultures were used to inoculatethe next stage. Media was prepared using the three batches of PP3 at 50g/L. One of the two batches that had resulted in lower biomassconcentrations of C. histolyticum during 5 L experiments (lot#5332398)was also prepared at 100 g/L.

FIG. 59 shows the results from the small scale experiment. It can beobserved that lot 5325635 and 5332398 showed reduced optical densities(600 nm) in the third stage of approximately 2.5 units, these weredeemed to be “poor” batches of PP3. Lot 5354796 maintains an opticaldensity (600 nm) of 5 units in the third stage of cultivation, this wasdeemed to be a “good” batch of PP3. Interestingly when the concentrationof a “poor” batch of PP3 (5332398) was increased to 100 g/L the sameoptical density (600 nm) was achieved in the second and third stage ofthe cultivation. This data does support the theory that the deviationsin growth profiles are caused by variation in the quantity of a limitingnutrient between batches of PP3. It was not possible to identify thisnutrient by analytical testing of the batches of PP3.

Evaluation of Proteose Peptone #3 at 100 g/L in 5 L Fermentation

The results of the small scale study demonstrated that increasing theconcentration of PP3 from 50 to 100 g/L removed the issue of batch tobatch variability. This process change was tested at 5 L scale using a“good” and “poor” batch of PP3 (lot 5354796 and 5325635, respectively)as determined during the small scale investigation into PP3 variability.FIG. 60 shows the growth profiles of the two fermentations. The twocultures show identical specific growth rates during the exponentialphase. The fermentation enter stationary phase and reach very similarmaximal optical densities (600 nm) of approximately 6.5 units. This datademonstrates that increasing the concentration of PP3 alleviates theissue of batch to batch variability of the PP3. Due to the higherbiomass concentration achieved and longer exponential phase in thefermentation harvest point was extended to 12 hours.

FIGS. 61 and 62 show SDS-PAGE analysis of the two fermentationsutilising 100 g/L PP3. The gels demonstrate consistent expression ofcollagenase in both fermentations. The samples from both fermentationsappear to contain similar levels of contaminant described in FIG. 56,although PBFT70d appears contain slightly more of the 40 kDa band(clostripain). It is possible that these small differences are due tostaining or loading differences. Again the quantity of clostripainproduced using the PP3 process is significantly lower than the Phytoneprocess. The precursor band appears to persist longer into the timecourse of the fermentation. It was recommended that future fermentationsat 100 g/L should be extended to a 14 hour harvest.

The presence of the precursor band highlights the importance of theharvest point definition and its qualification during processvalidation.

FIG. 63 displays data from densitometry analysis of the gel in FIG. 61.The chart compares product and precursor formation (densitometry peakarea) to cell growth (OD600). Product formation appears to be consistentwith cell growth and the rate of production decreases as the cultivationenters stationary phase. The precursor band decreases in intensity asexponential growth ends but is still present at the harvest point of thefermentation.

Scale-Up of 100 g/L Proteose Peptone #3 Fermentation to 200 L.

Following the increase in the PP3 concentration to 100 g/L the processwas scaled to 200 L. To generate the required quantity of inoculum forthe 200 L vessel a third inoculum stage was introduced using a 15 Lworking volume fermenter. 3×200 mL cultures were used to inoculate the15 L fermenter and following 12 hours of growth 8 L of the 15 L wereinoculated into the 200 L vessel. FIG. 64 compares the growth curve ofthe 200 L fermentation to the two 5 L fermentation using 100 g/L PP3. Asrecommended the growth profile was extended to 14 hours to ensure thatthe precursor band had completely disappeared before processing began.The growth profile of the 200 L fermentation is very similar to thefermentation at 5 L scale, demonstrating successful scale up of thecultivation.

FIG. 65 shows SDS-PAGE analysis of the time course of the 200 Lfermentation. The gel shows product formation during the course of thefermentation. The material at the 14 hour harvest point contains nodetectable pre-cursor and very low levels of contaminants. The productgenerated from the 200 L fermentation appears very similar to thatproduced from the 5 L process, indicating that the increased generationnumber of the 200 L process has not had a detrimental effect. FIG. 66displays data from densitometry analysis of the gel in FIG. 64. Thechart compares product and precursor formation (densitometry peak area)to cell growth (OD600). Product formation appears to be consistent withcell growth and the rate of production decreases as the cultivationenters stationary phase. The precursor band decreases in intensity asexponential growth ends. The precursor band decreases in intensity morerapidly in the 200 L fermentation than the 5 L cultivation, PBFT70c(FIG. 63). FIG. 67 shows SDS-PAGE analysis using a 4-12% Bis-Tris gel onthe 200 L fermentation time course. The approximate molecular weights ofthe detected contaminants are annotated on the gel.

The harvest process (clarification by filtration) developed for Process2 was evaluated during the 200 L scale up fermentation. The cell culturewas successfully clarified using the existing process with no blockageof the filter train. The harvest process is described in the materialsand methods section. 20 L of filtrate from the 200 L fermentation wasprocessed by DSP to demonstrate a partial scale up of the downstreamProcess 3 (infra).

Quantification of Product Yield by Densitometry Analysis

A more accurate and quantifiable method was required to determineproduct concentration during the upstream process step than thesemi-quantitative SDS-PAGE analysis (FIGS. 62 and 63). The fermentationfiltrate has a high quantity of pigment and peptides from the growthmedium that makes standard protein quantification techniques such as UVand the Bradford assay unusable. The semi-quantitative analysis carriedout previously was modified and updated by carrying out densitometryanalysis of the Coomassie stained gels. The method involved loading arange of quantities (0.2-1.2 μg/lane) of mixed AUXI and AUXII referencematerial and dilutions of the sample to be quantified onto a TrisGlycine gel. The scanned image was then analysed and the peak area forestimated for the standards and the samples. A standard curve was thenconstructed (total collagenase) and used to quantify the amount of totalcollagenase in the sample dilutions. FIG. 68 shows an example of acollagenase standard curve and highlights the linearity of thequantification method within the anticipated range of the samples. TheTris Glycine gels did not completely resolve AUXI and AUXII thereforethe total collagenase was quantified rather than attempting toseparately quantitate the two proteins.

The quantity of collagenase was analysed for PBFT70c, PBFT70d and the200 L scale-up fermentations. The quantity was found to be ˜280-350 mg/Ltotal collagenase for all three fermentations.

Materials and Methods Media Preparation: 1 L Media Preparation

The phosphates for the inoculum preparation (table 25) were autoclavedin a 1 L bottle at 121° C. for 20 minutes. The bulk media (table 26) wasinitially heated in a microwave to 60° C. to fully dissolve componentsbefore autoclaving in a 1 L bottle at 121° C. for 20 minutes. The PSA 1(table 27) was filtered through a 0.2 μm Sartopore 2 150 cm² filter intoa 250 mL sterile bottle. The 300 mL autoclaved phosphates, 600 mLautoclaved bulk media and 100 mL sterile filtered PSA 1 were pooledbefore aliquoting into 30 mL gamma irradiated universals (8×5 mL) and500 mL Erlenmeyer flasks (4×200 mL).

TABLE 25 Phosphate composition for inoculum preparation ComponentQuantity Required KH₂PO₄ 1.92 g K₂HPO₄ 1.25 g Na₂HPO₄ 3.5 g NaCl 2.5 gDeionised Water Up to 300 mL

TABLE 26 Bulk medium composition for inoculum preparation ComponentQuantity Required Proteose Peptone # 3 50 g or 100 g* Yeast Extract 8.5g Deionised Water Up to 600 mL *Medium recipe includes PP3 at 50 and 100g/L.

TABLE 27 PSA 1 Magnesium/Glucose composition for inoculum preparationComponent Quantity Required MgSO₄ × 7H₂O 0.08 Glucose 5 g Vitaminsolution 10 mL Deionised Water Up to 100 mL

TABLE 28 Vitamin solution for inoculum preparation Component QuantityRequired FeSO4 × 7H2O 1.2 g Riboflavin 50 mg Niacin 100 mg CalciumPantothenate 100 mg Pimelic acid 100 mg Pyridoxine 100 mg Thiamine 100mg Deionised Water Up to 1 litre

5 L Media Preparation

The phosphate solution for the 5 L scale (table 29) was autoclaved in a1 L bottle at 121° C. for 20 minutes. The bulk medium (table 30) wasadded directly to the 5 L vessel and autoclaved at 121° C. for 20minutes. The PSA 1 (table 31) was filtered through a 0.2 μm Sartopore 2150 cm² filter into a 500 mL sterile bottle. The 250 mL phosphatesolution and 200 mL PSA 1 was separately pumped into the 5 L vessel oncompletion of autoclaving and cooling of the vessel.

TABLE 29 Phosphate composition for 5 L fermentation Component QuantityRequired KH₂PO₄ 9.22 g K₂HPO₄   6 g Na₂HPO₄ 16.8 g NaCl   12 g DeionisedWater Up to 250 mL/278.35 g

TABLE 30 Bulk medium composition for 5 L fermentation Component QuantityRequired Proteose Peptone #3 240 g or 480 g* Bacto Yeast Extract 40.8 gDeionised Water Up to 4.35 L *Medium recipe includes PP3 at 50 and 100g/L.

TABLE 31 PSA 1 Magnesium/Glucose composition for 5 L fermentationComponent Quantity Required MgSO₄ × 7H₂O 0.38 g Glucose 24 g Vitaminsolution 48 mL Deionised Water Up to 200 mL/200 g

TABLE 32 Vitamin solution for 5 L fermentation Component QuantityRequired FeSO4 × 7H2O 1.2 g Riboflavin 50 mg Niacin 100 mg CalciumPantothenate 100 mg Pimelic acid 100 mg Pyridoxine 100 mg Thiamine 100mg Deionised Water Up to 1 litre

15 L Media Preparation

The phosphate solution (table 33) was filtered through a 0.2 μmSartopore 2 300 cm² filter into a sterile 2 L bottle. The bulk medium(table 34) was added directly to the 20 L vessel prior to Steam-In-Place(SIP) sterilisation of the vessel. The PSA 1 (table 35) was filteredthrough a 0.2 μm Sartopore 2 300 cm² filter into a 1 L sterile bottle.The 750 mL phosphates and 600 mL PSA 1 were separately pumped into the20 L vessel on completion of SIP and cooling of the vessel.

TABLE 33 Phosphate composition for 15 L fermentation Component QuantityRequired KH₂PO₄ 27.66 g   K₂HPO₄ 18 g Na₂HPO₄ 50.4 g   NaCl 36 gDeionised Water Up to 750 mL/835.05 g

TABLE 34 Bulk medium composition for 15 L fermentation ComponentQuantity Required Proteose Peptone #3 1.44 kg Bacto Yeast Extract 122.4g Deionised Water Up to 13.05 L

TABLE 35 PSA 1 Magnesium/Glucose composition for 15 L fermentationComponent Quantity Required MgSO₄ × 7H₂O 1.14 g Glucose 72 g Vitaminssolution 144 mL Deionised Water Up to 600 mL/600 g

TABLE 36 Vitamin solution for 15 L fermentation Component QuantityRequired FeSO4 × 7H2O 1.2 g Riboflavin 50 mg Niacin 100 mg CalciumPantothenate 100 mg Pimelic acid 100 mg Pyridoxine 100 mg Thiamine 100mg Deionised Water Up to 1 litre

200 L Media Preparation

The phosphate solution (table 37) was filtered through a 0.2 μmSartopore 2 300 cm² filter into a Gammasart Biosystem SA10 10 L bag. Thebulk media (table 38) was added directly to the 200 L vessel prior toSIP sterilisation of the vessel. The PSA 1 solution (table 39) wasfiltered through a 0.2 μm 300 cm² filter into a Gammasart Biosystem SA1010 L bag. The 10 L phosphates and 8 L PSA 1 were separately pumped intothe 200 L vessel on completion of SIP and cooling of the vessel.

TABLE 37 Phosphate composition for 200 L fermentation Component 4 ×Fermenters KH₂PO₄ 368.8 g   K₂HPO₄ 240 g Na₂HPO₄ 672 g NaCl 480 gDeionised Water Up to 10 L/11.134 kg

TABLE 38 Bulk medium composition for 200 L fermentation ComponentQuantity Required Proteose Peptone #3 19.2 kg Bacto Yeast Extract 1.632kg Deionised Water Up to 174 L

TABLE 39 PSA 1 Magnesium/Glucose composition for 200 L fermentationComponent Quantity Required MgSO₄ × 7H₂O 15.2 g Glucose 960 g Vitaminssolution 1.92 L Deionised Water Up to 8 L/8 kg

TABLE 40 Vitamin solution for 200 L fermentation Component QuantityRequired FeSO4 × 7H2O 2.4 g  Riboflavin 100 mg Niacin 200 mg CalciumPantothenate 200 mg Pimelic acid 200 mg Pyridoxine 200 mg Thiamine 200mg Deionised Water Up to 2 L/2 kg

Fermentation

FIG. 69 illustrates overviews of the process flows for the Phytone andPP3 fermentation processes at 5 and 200 L scale.

5 L Scale Fermentation

A vial of the WCB (2005#1019D) was thawed and 50 μL aliquots were usedto binoculate 8×5 mL of inoculum media in 30 mL gamma irradiateduniversals. The 5 mL cultures were incubated at 37° C. in an anaerobicjar in the presence of 3 anaerobic gas packs. After approximately 12hours of incubation (OD600 3.0-4.0) 2×5 mL cultures were selected andused to inoculate 2×200 mL inoculum media in 500 mL Erlenmeyer flasks.The two flasks were placed together in an anaerobic jar with 3 gas packsand were incubated at 37° C. in a shaking incubator (70 rpm) for 12hours. After 12 hours of incubation (OD600 6.0-7.0) each 200 mL inoculumwas used to inoculate a 5 L vessel.

The working volume of the 5/7 L vessels FT Applikon vessels was 5 L ofwhich 4% (v/v) was inoculum from the 200 mL stage. The agitation ratewas set at 100 rpm. The pH, dO2 and temperature were controlled at 7.00units, 0% of saturation and 37° C. respectively. The pH was controlledwith additions of either HCl (5M) or NaOH (5M). The dO2 concentrationwas maintained at 0% by continuous sparging of nitrogen, with a flowrateof 1 L/min. Samples were taken during the fermentation and filteredthrough 0.2 μm filters before storing at −20° C. for analyticalpurposes. The fermentations began to enter stationary phase at an OD600of 6.0-7.0. After 12 hours the fermenter was cooled to 10-20° C. beforecommencing harvest recovery.

200 L Scale Fermentation

A vial of the WCB (2005#1019D) was thawed and 50 μL aliquots were usedto inoculate 8×5 mL of inoculum media in 30 mL gamma irradiateduniversals. The 5 mL cultures were incubated at 37° C. in an anaerobicjar in the presence of 3 anaerobic gas packs. After approximately 12hours of incubation (OD600 3.0-4.0), 4×5 mL cultures were selected andused to inoculate 4×200 mL inoculum media in 500 mL Erlenmeyer flasks.Two flasks were placed together in anaerobic gas jars with 3 gas packsand left to incubate at 37° C. in a shaking incubator (70 rpm) for 12hours. After 12 hours of incubation (OD600 6.0-7.0) three of the fourflasks were pooled together and used to inoculate the 20 L vessel.

The working volume of the 20 L vessels was 15 L of which 4% (v/v) wasinoculum from the 200 mL stage. The agitation rate was set at 100 rpm.The pH, dO2 and temperature were set at 7.00 units, 0% and 37° C.respectively. The pH was controlled with additions of either HCl (5M) orNaOH (5M). The dO2 concentration was maintained at 0% by continuousheadspace sparging of nitrogen, with a flowrate of 20 L/min.

After 12 hours of growth in the 20 L vessel (OD600 6.0-7.0), 8 L ofculture were used to inoculate the 200 L vessel. The running conditionswere identical to the 20 L scale. The final optical density (600 nm) atharvest was 6.0-7.0. After 14 hours the fermenter was cooled to 10-20°C. before commencing harvest recovery.

Harvest 5 L Harvest

The 5 L cultures were pumped with a flow rate of 5 L/h through aMillistak+10″ Opticap depth filter (Millipore, KC0HC10FF1) and 0.2 μmSartopore 2 300 cm² filter into sterile 250 mL bio-containers. Theprocessed material was either stored at −20° C. or stored at 4° C.overnight before processing by DSP.

200 L Harvest

The 200 L harvest was performed using a filtration harvest train. Theculture was pumped with a flow rate of 200 L/h through aMilistak+(MC0HC10FS1) disposable depth filter with a filtration area of4×1 m2 followed by two 0.2 μm Express Opticap XL filters, 2×0.49 m2(Millipore, KHGES10TT1). The process time for primary clarification was1 hour. An additional 10 min was allowed at the end of the harvest toretrieve residual product held up in the filters. The clarifiedsupernatant was collected in a 200 L Stedim Palletank with the filtrateweight recorded. 20 L of filtrate was passed through a MUSTANG Q highaffinity DNA column with a flowrate ˜6 L/min and collected into twosterile 20 L stedim bags, prior to storage at 4° C. overnight.

Analysis Optical Density Measurements

The spectrophotometer was blanked using PBS at wavelength 600 nm.Fermentation samples were diluted by factors of 10, 20 or 100 (dependenton cell density) using PBS. 1 mL of each diluted sample was transferredinto a 1 mL cuvette; the top was sealed and inverted 5 times beforerecording triplicate optical density readings at a wavelength of 600 nm.

Tris-Glycine Gels

Fermentation samples were filtered through 0.2 μm filters beforepreparing them for SDS-PAGE analysis. 10 μl of filtered sample was addedto 10 μl sample buffer (2×), 2.5 μl reducing agent (10×) and 2 μl of0.1M EDTA (to achieve final concentration of 10 mM). The high molecularweight (HMW) marker was prepared by adding 10 μl of concentrated stockto 80 μl reducing agent (10×), 310 μl WFI and 400 μl sample buffer (2×).The diluted HMW standard was then heated to 95° C. for 5 minutes beforealiquoting and storage at −20° C. for use in subsequent gels. 15 μL offermentation sample and 10 μL of HMW marker were run on 8% Tris-Glycinegel using pre-cooled (4° C.) Tris-Glycine running buffer at 130V, 400 mAand 100 W for ˜1 hour and 50 minutes. After electrophoresis, the gelswere immersed in 100 mL colloidal blue stain reagent (55 mL WFI, 20 mLmethanol, 5 mL stainer A, 20 mL stainer B) and left to stain for 5 h onan orbital shaker at 60 rpm. Gels were de-stained with 200 mL WFI. Thegel was left in WFI for 15-20 h until excess stain was removed afterwhich the gel was scanned and dried according to the manufacturesinstructions.

Bis-Tris Gels

The fermentation samples were prepared for SDS-PAGE analysis by adding10 μl of 0.2 μm filtered sample to 4 μl sample buffer (4×), 1.5 μlreducing agent (10×) and 1.7 μl of 0.1M EDTA (to achieve finalconcentration of 10 mM). 15 μL of fermentation sample and 10 μL of Mark12 marker were run on a 4-12% Bis-Tris gel and run using MES runningbuffer at 200V, 400 mA and 100 W for ˜40 mins. After electrophoresis,the gels were immersed in a 100 mL fixing solution (40 mL dH2O, 50 mLmethanol, 10 mL acetic acid) for 10 minutes before replacing with a 95mL staining solution (55 mL dH2O, 20 mL methanol, 20 mL stainer A) for afurther 10 minutes. 5 mL of stainer B was added to the staining solutionand the gels were left to stain for 5 h on an orbital shaker at 60 rpmbefore de-staining with 200 mL WFI. The gel was left in WFI for 15-20 huntil excess stain was removed after which the gel was scanned and driedaccording to the manufactures instructions.

Process 3 Purification:

The first 20 L scale run-through of a newly developed process (Process3) for the purification of collagenases from Clostridium histolyticum,which was modified from Process 2 performed to GMP at 20 L scale.Significant process changes were introduced in the development ofProcess 3 in order to make the purification more robust and moreamendable to scale up and subsequent process validation. One significantfactor in facilitating this process change was in the choice offermentation component. Process 2 had been based on the requirement tomaintain a phytone based fermentation media whereas for process 3proteose peptone No. 3 was use. The process run-through is split intothe key steps of the down stream purification and the collagenases AUXIand AUXII. These include the treatment of the fermentation filtrateusing a MUSTANG Q capsule, hydrophobic interaction chromatography,tangential flow filtration step 1 (denoted TFF1), anion exchangechromatography and tangential flow filtration step 2 (denoted TFF2).AUXI and AUXII co-purify in the initial steps of the purification andare only separated during the anion exchange chromatography step(performed using QSepharose HP media). AUXI and AUXII are then processedseparately and formulated. The intermediates are then mixed in a 1:1ratio (based on protein content determined by UV) and filtered to formthe drug substance. In developing process 3, key steps associated withprocess 2 were removed. Notably the ammonium sulphate precipitationstep, two chromatography steps (hydroxyapatite and gel permeationchromatography) and all −20° C. hold steps were eliminated. The use ofun-scaleable steps such as stirred cells and dialysis were also removedand replaced with tangential flow filtration (TFF). The issue of productinstability, which was evident in process 2 (and eliminated the use ofTFF), was not apparent in the 20 L scale run of process 3. Thecontaminant profile associated with process 3 was however different toprocess 2 in which clostripain and gelatinase had been major components.Most notably a 40 kDa, 55 kDa and two 90 kDa contaminants (oneco-purifying with AUXI and the other with AUXII) were detected bySDS-PAGE. As a result of these new contaminants, some of the QC assays(such as RPHPLC and SEC-HPLC) were of limited use since they did notresolve all process 3 impurities. The inability to utilize establishedQC assays for in-process purity determination, resulted in the need todefine a method for establishing which material form the QSepharosecolumn was suitable for further purification. This was required sincethe contaminants were not clearly resolved from the AUXI and AUXIIproducts on the QSepharose column and it was therefore necessary tocollect eluted material in discrete fractions, which could be analyzedretrospectively. Analysis was performed by SDS PAGE and the poolingdecision for the 20 L run-through was based on experience of therelative staining intensity of impurity to product using a standardized1 μg load.

Retrospective densitometry analysis of SDS-PAGE enabled the poolingcriteria to be described based on relative percent product purity.Further densitometry analysis using material from the 200 Ldemonstration run enabled a standardized method to be established aswell as an approximation of assay variation. This led to an agreedprocedure for the pooling of in-process fractions to be implemented inthe first GMP campaign.

In addition to the process description, preliminary work describing abuffer stability and in-process sample stability study is presentedalong with initial characterization of some of the impurities associatedwith Process 3.

Process 3 differed from process 2 in three main areas. Firstly, theammonium sulphate precipitation step and hydroxyapatite chromatographysteps were removed; secondly, the gel permeation chromatography (GPC)step was eliminated and thirdly, all buffer exchange steps wereperformed by tangential flow filtration. The precipitation step wasreplaced by the use of hydrophobic interaction chromatography (HIC) atthe client's recommendation. Development of this step resulted in thesuccessful implementation of HIC for (i) product capture (therebyserving as a concentration step); and (ii) some protein and pigmentcontaminant removal. The HIC step was also subsequently shown to reducelevels of dsDNA. As a result of the process development program, theintroduction of HIC and inclusion of a MUSTANG Q filter step removed theneed for both the ammonium sulphate precipitation step and thehydroxyapatite chromatography step. The overall effect was to simplifythe up front capture of product and to remove a potential hold stepassociated with Process 2. This latter point had significance in thatpreviously the fermentation could be assessed prior to down streampurification since the pellets resulting from the precipitation stepcould be held at −20° C. prior to processing.

Following the HIC step, product was buffer exchanged using tangentialflow filtration (TFF). This was performed using 30 kDa molecular weightcut off (MWCO) membranes and replaced the dialysis procedure used forProcess 2. Aggregate contamination, which when present was detected asAUXII-derived, appeared to be removed during the anion exchangechromatography step (IEX). As a result, the GPC step was eliminatedsince both AUXI and AUXII intermediates were within specification foraggregates following IEX. Finally, the final concentration andformulation of the AUXI and AUXII intermediates was performed using TFFinstead of the previous method of utilizing stirred cells.

Overall, Process 3 represented a simpler process that was more amenableto scale up and validation than Process 2. In addition, the reduction inconsumable cost was apparent by the elimination of the need forhydroxyapatite and gel permeation media and by the reduced number ofsteps requiring leupeptin. An overview of the purification scheme forProcess 3 is given in FIG. 46.

Non-GMP Demonstration Run at 20 L Scale

Process 3 was performed at 20 L scale in the process developmentlaboratories in order to demonstrate if material of suitable qualitycould be generated using this modified process at 20 L scale. A keyrequirement for processing was the ability to limit potential proteaseactivity by performing steps chilled wherever possible and by theinclusion of the cysteine protease inhibitor leupeptin at key stages inthe procedure. A full 20 L of fermentation filtrate was processed sincethe feedstock was generated from 200 L fermentation PP3. Details of thefermentation and subsequent harvest and filtration are documented in aseparate report.

MUSTANG Q Filter Treatment of Fermentation Filtrate

Following 0.2 μm filtration, approximately 22 L of fermentationsupernatant was loaded onto a MUSTANG Q chromatography capsule asdescribed previously. Some visible pigment contamination (green/brown)appeared to be removed by the MUSTANG Q capsule during the filtration ofthe first 10 L since the contents of the first 10 L Stedim bag appearedvisibly less pigmented than the second. The ability of the MUSTANG Qcapsule to remove dsDNA was monitored across this step by pico greenanalysis of pre and post MUSTANG Q filter samples (Table 41). In processanalysis indicated that unlike previous data generated at small-scale,bulk nucleic acid removal was not evident at the MUSTANG Q filter step.The robustness and application of this step therefore requires furtherinvestigation.

TABLE 41 Sample description Result ng/ml Fermentation filtrate 230.65Post Mustang Q 216.53 Post HIC 1.02 Post TFF 6.34 Post IEX Aux I 2.33Post IEX Aux II 3.41

Hydrophobic Interaction Chromatography (HIC)

The use of HIC served three functions in the purification. Firstly, theproduct was reduced in volume since conditions were identified in whichcollagenases bound to the resin. Secondly, some pigment and proteincontaminant was removed at this stage and thirdly, pico green analysisfrom this run indicated reduction of dsDNA. The HIC step was performedusing supernatant processed directly from the fermentation (afterMUSTANG Q treatment) and, as a result a hold step, (evident in Process 2as the ammonium sulphate pellet) was no longer present for Process 3.

In order to provide conditions for collagenases to bind to the HICcolumn, product (20 L) from the MUSTANG Q filter step was diluted with a3M-ammonium sulphate solution to a final concentration of 1M. Afterfiltration, product was loaded onto the column and eluted using a 2-stepisocratic elution.

The protein concentration of the HIC load material was difficult todetermine accurately and was estimated in two ways. Firstly, a Bradfordassay was performed on the material prior to ammonium sulphate addition.This was performed with undiluted material in order to standardize thecontribution from pigment present in the fermentation media, which wasknown to interfere with the assay. Secondly, the estimate was based onvolume of fermentation media loaded per mL of column resin. The columnload was estimated to be 5.9 mg of total protein/mL resin by Bradfordassay or alternatively ˜13 mL of fermentation media per mL of resin. Anestimate of the total amount of target protein eluted from the columnwas determined as 3.4 g using UV (see Table 42). Assuming that the totalprotein present in the HIC load was 9 g (Bradford assay), this equatedto a 38% recovery. This value was only regarded as a relative measure,however, due to the inaccuracy of the assay for the samples containingfermentation media components.

An alternative method for estimating the HIC load concentration wasdetermined using densitometry although it was recognized that thisestimation would give a collagenase content rather than estimate oftotal protein (which could vary between fermentations). Using thisapproach, the total collagenases were estimated as 360 mg/L with anapproximate ratio of AUXI to AUXII estimated as 40:60. Using this data,the total collagenase expected in the HIC load would be 7.2 g giving astep yield of 47%.

The chromatogram resulting from the HIC step is shown in FIG. 70.Visible pigment was apparent in the flow-through as well as bound to thecolumn. After washing the column with equilibration buffer to remove theflow-through contamination, peak 1 was eluted using an intermediateconcentration ammonium sulphate solution (0.3M).

This peak was shown to contain protein contaminants although some AUXIIwas also eluted at this stage (FIG. 71). This loss in product wasexpected and had been noted previously. In order to minimize the amountof product lost, without compromising purity, the elution volume forpeak 1 removal was set at 5 column volumes. Peak 2, containing themajority of the product, was then eluted using buffer with no ammoniumsulphate. Peak 2 was collected as a single pool with the chromatographymethod programmed so that collection began after ¾ of a column volume ofelution buffer had been applied to the column.

Collection was then terminated after a total of 4 column volumes hadbeen collected. In order to minimise potential proteolysis in theproduct at this stage in the process, leupeptin was added to the postHIC eluate and the material held at 2-8° C. The hold time for the postHIC eluate was of 2 day duration.

Tangential Flow Filtration 1 (TFF1)

TFF using 30 kDa membranes was introduced following the HIC in order toreduce the volume of product (5-fold) and to exchange the buffer intoconditions suitable for binding to the anion exchange column. Ofparticular importance was the sufficient reduction in ammonium sulphatesuch that the conductivity of the IEX load sample was <1.8 mS. Thediafiltration buffer was chilled and leupeptin added prior to use toreduce the likelihood of proteolysis. No loss in protein was estimatedover the course of this step (>100% recovery) although this may reflectthe inaccuracy in protein concentration estimation at this stage in theprocess due to the presence of pigment in the pre TFF1 material.Approximately 97.5% of the total protein (3325 mg) was recovered in theretentate with an additional 204.8 mg recovered in the first membranerinse (infra). Filtration of the total protein from the combinedretentate and rinse was performed at the end of the TFF step prior toholding the material overnight at 2-8° C. SDS-PAGE analysis indicated nosignificant differences were detected before and after the TFF step(FIG. 71).

Q-Sepharose Chromatography

The Q-Sepharose column was loaded at a maximum capacity of 5 mg totalprotein per mL resin. As a result, not all of the available materialfrom the TFF step was utilized in this step (see Table 421). TheQ-Sepharose column resolved AUXI and AUXII collagenases as expected(FIG. 72). The start of AUXII elution began at approximately 13.6% B(where buffer A=10 mM Tris, 0.2 mM leupeptin pH 8 and buffer B=BufferA+360 mM NaCl) which equated to a post column conductivity of 5.7 mS.Fractions (100 mL) were collected throughout the elution of AUXII untilthe absorbance value dropped to 25% of the peak height (550 mAU). Asmall peak was eluted at approximately 8 mS (20.3% B) following AUXIIelution. In-process analysis of this peak from previous small-scaleexperiments indicated this to be AUXII derived aggregate material. Thestart of AUXI elution was at approximately 27% B (which equated to 10.4mS). As before, 100 mL fractions were collected until the absorbancedropped to the required 25% value (190 mAU).

Each AUXI and AUXII fraction collected was analyzed by SDS-PAGE andsubjected to densitometry (FIGS. 73-76). Densitometry was performedretrospectively, so the decision on fraction pooling was based onexperience of the levels of contaminant visible by Colloidal bluestaining. In consultation with Auxilium, fractions 6-12 were pooled forthe AUXII product and fractions 19-26 pooled for AUXI. The step yieldsand protein concentrations present in the material pooled from theQ-Sepharose run are included in table 42.

SDS-PAGE analysis of the post IEX AUXI and AUXII products from the 20 Ldemonstration run (FIGS. 77 and 78) showed few contaminants visible bySDSPAGE. In addition, the contaminants detected were in accordance withprevious small-scale experiments although there were noted differencesin the resolution of the contaminants, which appeared to be more defined(i.e. separate peaks or shoulders) in the small-scale model. Thesecontaminants were also different to those identified for Process 2 inwhich clostripain and gelatinase had been major components. As a result,the QC protocols developed for Process 2 were not optimized for thedetection of the new contaminants associated with Process 3.

Retrospective densitometry of the pooled material estimated the purityat 95.1% for AUXI and 99.4% for AUXII. Currently, however the purityspecification of ≧97% is specified by RP-HPLC and no final productspecification has been established using densitometry.

Concentration and Buffer Exchange of AUXI and AUXII

The separated AUXI and AUXII products from the Q Sepharose column wereprocessed separately by TFF using a 30 kDa membrane. This step wasrequired to: (i) remove/reduce leupeptin in the final product; (ii)formulate the intermediates into the correct buffer (10 mM Tris, 60 mMsucrose pH 8); and (iii) to achieve the required target proteinconcentration of 0.9-1.1 mg/mL. A total of 799 mg (˜683 mL at 1.17mg/mL) of AUXII and 860 mg (796 mL at 1.08 mg/mL) of AUXI wasconcentrated to a target concentration of 1.75 mg/mL. This theoreticalconcentration was based on the calculated reduction in volume requiredassuming no loss of product during the concentration step. Diafiltrationwas then performed into the required formulation buffer, the membraneswashed with the minimum volume of the TFF system (˜250 mL) and the fullamount combined with the concentrate to achieve the required targetconcentration of 0.9-1.1 mg/mL. A total of 819.5 mg AUXII (at 1.03mg/mL) and 797.0 mg of AUXI (at 1.09 mg/mL) were available afterfiltration. In both cases, the majority of product was recovered in theretentate and was estimated as 95.4% (762 mg) for AUXII and 83.1% (715mg) for AUXI. The additional material provided by the membrane rinse wasestimated as 153 mg and 89.6 mg for AUXII and AUXI respectively.

Mixing of Intermediates to Drug Substance

Approximately 200 mg of each intermediate was combined to give 400 mg ofthe drug substance. This was then filtered and approximately 26 mgprovided to QC for testing. The QC results for AUXI, AUXII intermediatesand the drug substance are provided in Table 43. All tests on the drugsubstance and AUXII intermediate passed the required specification. Thetest for potency of the intermediate AUXI however, was not within thespecified range although all other tests passed. With the exception ofthe AUXI potency result, these data indicated that Process 3 was capableof generating material of the required specification when purified atthe 20 L scale.

As well as QC testing, material from the 20 L demonstration run wasutilized for method validation at KBI BioPharma, Inc. At the client'srequest, 200 mg of drug substance was shipped on dry ice to KBI for drugsubstance and drug product methods validation. The latter testing wasperformed after lyophilisation of the drug substance at KBI. Inaddition, 25 mg of each intermediate was supplied to KBI for validationof analytical methods.

The individual step yields for the 20 L demonstration run are given intable 42. An extrapolation of the data in which all the availablematerial had been loaded onto the Q-Sepharose column indicated that themaximum total amount of available drug substance from this processrun-through was 1.6 g (assuming no loss of material through retains).This equates to an approximate overall process yield of 17.8% based onthe initial estimate of 9 g (using the Bradford assay) for the amount oftotal protein available to load onto the HIC column. With the limitationon the load for the Q-Sepharose column, a maximum of 1.4 g of drugsubstance was available from the current run-through if all theavailable intermediate had been mixed to form the drug substance.

TABLE 42 Total Protein conc. Amount protein Process step (mg/mL)(weight/volume) (mg) Step yield Fermentation — 200 L — — Pre Mustang Q —22 L Post Mustang Q 0.45 22 L 9000 — (Bradford) HIC load  0.30* 30 L9000 100 (theoretical) HIC peak 2 0.56 6101.7 mL 3416.95 38 (afterleupeptin) Pre-TFF1 0.54 6317.7 mL 3411.6 100 Post TFF1 2.55 1378.5 g3515.2 >100% (with wash 1 and post filtration) Q load 2.55 1216 mL3100.8 100 (5 mg/mL resin) Q AUXII pool 1.17 682.8 mL 798.88 25.8 Q AUXIpool 1.08 796.4 mL 860.11 27.7 Pre TFF2 1.17 682.80 mL 798.88 100 AUXIIIntermediate 1.03 795.6 g 819.47 >100 AUXII (post filtration) Pre TFF21.08 796.40 mL 860.11 100 AUXI Intermediate 1.09 731.2 g 797.01 92.7AUXI (post filtration) *calculated based on dilution factor afterammonium sulphate addition

TABLE 43 Drug substance Intermediate AUXI Intermediate AUXII Test Method(AXS2006A0754H) (AXS2006A0745H) (AXS2006A0737H) Appearance of SolutionQC SOP Clear, colourless with 2-3 Clear, colourless and free Clear,colourless with 2-3 001 small exogenous fibres from Particulate mattersmall exogenous fibres 1 mm in length (AK/1573/121) 1 mm in length(AK/1573/121) (AK/1573/121) Potentiometric QCSOP 7.6 Not required Notrequired measurement of pH 002 (FR/1598/098) Endotoxin QCSIO <0.5 EU/mL<0.5 EU/mL 0.136 EU/mL Determination 018 (AS/1597/128) (AS/1597/128)(AS/1597/128) Identity by SDS-PAGE QC SOP Major collagenase bands Majorcollagenase bands Major collagenase bands Coomassie Stained 103 between107 and 110 kDa between 110 and 115 kDa between 107 and 110 kDa and 110and 115 kDa (AS/1597/133) (AS/1597/133) (AS/1597/133) Total Protein byQC SOP 1.04 mg/mL 1.11 mg/mL 0.90 mg/mL Absorbance 144 (AS/1597/106)(AS/1597/106) (AS/1597/106) Spectrophotometry Rat Tail Tendon QC SOP2326 1483 Not Required Collagen Assay for 105 (1700-3500) (1900-3300)Potency (AS/1597/124) (AS/1597/124) (OOS/Keele/2006/0038) Reference 20972097 (2014-3440) (2014-3440) Potency for Class II QC SOP 57677 GPAunits/mg Not Required 119552 GPA units/mg Collagenases 106 (50000-90000)(79000-170000) (AS/1597/111) (AS/1597/111) Reference 69523 GPA units/mg69523 GPA units/mg (58000-95000) (58000-95000) Host Cell Protein AssayQCSOP <LOD Not Required Not Required 107 (FR/1589/108) Host Cell DNAAssay External NewLab Not Required Not Required Analysis of proteins QCSOP 100% main peaks 100% main peak 100% main peak using the Agilent 1100109 (47.70% AUX-I, 0% aggregates 0% aggregates HPLC System 52.30%AUX-II) (AK/1573/122) (AK/1573/122) (Identity and purity by(AK/1573/122) size exclusion chromatography) Reference AUX-I 100% AUX-I100% AUX-II 100% AUX-II 100% Analysis of proteins QC SOP 48.89% AUX-I99.14% AUX-I 0.39% AUX-I using the Agilent 1100 109 50.99% AUX-II 0.37%AUX-II 99.35% AUX-II HPLC System (Identity 0.12% Others 0.49% Others0.26% Others and purity by reverse (AK/1573/125) (AK/1573/125)(AK/1573/125) phase liquid chromatography) Reference AUX-I 93.92%,AUX-II AUX-I 93.92%, AUX-II AUX-II 100% 4.96%, Others 1.13% 4.96%,Others 1.13% AUX-II 100% Analysis of proteins QC SOP 0.3% Gelatinase0.4% Gelatinase 0% Gelatinase using the Agilent 1100 109 (AK/1573/131)(AK/1573/131) (AK/1573/131) HPLC System (Gelatinase by anion exchangechromatography) Reference AUX-I 0% Gelatinase AUX-I 0% Gelatinase AUX-I0% Gelatinase AUX-II 0% Gelatinase AUX-II 0% Gelatinase AUX-II 0%Gelatinase Peptide Mapping by QC SOP Not Required Tuesday TuesdayTryptic Digest and 110 Reverse Phase HPLC Residual Leupeptin QCSOP Notdetected Not required Not required 141 <0.5% w/w (AK1573/136) BioburdenQCSOP 0 cfu/5 mL 0 cfu/5 mL 0 cfu/5 mL 223 (JM/1505/115) (JM/1505/114)(JM/1505/112)

Sample Stability Study

During the 20 L demonstration run, samples were taken at key processpoints. As the demonstration run was performed as a continuous process(with no hold steps) an attempt was made to assess the stability ofin-process material during the hold times anticipated for GMP batches.The extended run duration expected for GMP was recognized due to therequirement to obtain equipment clearance data between process steps.In-process material was held at 2-8° C. for approximately the durationexpected for the GMP manufacture. In addition samples were held for anextended time representing twice that expected for the GMP campaign. Adescription of the samples taken, along with the respective hold timesis given in table 44. The processing times for the 20 L demonstrationrun are represented in table 45. All samples were submitted to QC forSDS-PAGE, RP-HPLC, SEC-HPLC and UV analysis (FIGS. 79-83).

Overall, the results showed no detectable deterioration in the productover the first hold point with respect to purity (as determined byRP-HPLC), degradation (as detected by 8% Tris-Glycine SDS-PAGE) andaggregation (as determined by SECHPLC). Some of the assays, however,were recognised to be limiting since low molecular mass components wouldnot be detected by 8% SDS-PAGE and the RPHPLC assay had not beendeveloped to detected the 40 kDa, 55 kDa and 90 kDa contaminantsassociated with Process 3. Some assays were also less relevant for crudesamples such as the use of UV and SEC-HPLC in the fermentation samples.Despite these limitations, the only detected change in product profilewas identified for the second hold point (day 12) for the AUXIIin-process sample taken from the Q-Sepharose column. This showed anincrease in aggregate level between day 5 and day 12 although thisincrease was only from 0 to 0.62%.

A second stability study was performed on the in-process retains whichwere taken at the point of manufacture during the 20 L demonstration runand stored at −20° C. In this study, samples were thawed and incubatedat room temperature and at 37° C. and monitored by 4-12% SDS-PAGEanalysis to allow the full molecular mass range of contaminants to beevaluated (FIGS. 84-88). These data demonstrated that the samples priorto Q-Sepharose anion exchange were vulnerable to degradation. Followingseparation of the collagenases AUXI and AUXII (by the Q-Sepharosecolumn), the samples appeared to be relatively stable and lookedcomparable to the time zero samples by SDS-PAGE.

Taken together, both studies indicate that providing the temperature ismaintained between 2-8° C., in-process material is not expected todeteriorate during processing over the hold times investigated. Thisgives a level of confidence that the use of leupeptin and temperaturecontrol is sufficient to restrict levels of product degradation duringprocessing over the durations anticipated in GMP.

TABLE 44 Duration held at 2-8° C. before Storage, Sample freezing VolumeRetain Container Fermentation DAY 4 1 × 2 mL −70° C. Bag Filtrate DAY 51 × 2 mL Post Mustang Q DAY 4 1 × 2 mL −70° C. Bag Post HIC DAY 3 1 × 2mL −70° C. Bag DAY 6 1 × 2 mL Post TFF DAY 2 1 × 2 mL −70° C. Bag DAY 41 × 2 mL Post IEX AUX I DAY 5 2 × 1 mL −70° C. Biotainer DAY 12 2 × 1 mLPost IEX AUX II DAY 5 2 × 1 mL −70° C. Biotainer DAY 12 2 × 1 mL AUX IIntermediate DAY 6 2 × 1 mL −70° C. Biotainer DAY 12 2 × 1 mL AUX II DAY6 2 × 1 mL −70° C. Biotainer Intermediate DAY 12 2 × 1 mL

TABLE 45 Process step 20 L demonstration run Fermentation harvest Day 1Mustang Q Day 1 HIC Day 2 TFF1 Day 5 IEX (Q-Sepharose) Day 6 TFF2 (AUXI)Day 7 TFF2 (AUXII) Day 9 DS mixing Day 12

Buffer Stability Study

Buffer samples illustrated in table 46 were reserved from the 20 Ldemonstration and retested after storage at 2-8° C. The pH,conductivity, temperature and appearance of the buffers were noted atthe time of completion and after 12-13 day's storage. The results ofthis study are given in table 47. Small differences were observed in thevalues for pH and conductivity but this may be due to differences intemperature between the original buffers and the tested retains. Inparticular, the HIC buffers showed the largest variation in conductivityand temperature. As a result, future studies on buffer stability shouldinclude specification of an accepted temperature range for recording allparameters. In all cases, the buffer retains were clear in appearance attime zero and after the required hold time.

TABLE 46 BUFFER CONSTITUENTS HIC A 10 mM Tris, 1.0M Ammonium Sulphate,pH 8.0 HIC A2 10 mM Tris, 0.3M Ammonium Sulphate, pH 8.0 HIC B 10 mMTris, pH 8.0 DIAFILTRATION 10 mM Tris, 200 μm Leupeptin, pH 8.0 IEX A 10mM Tris, 3 mM CaCl₂, 200 μm Leupeptin, pH 8.0 IEX B 10 mM Tris, 3 mMCaCl₂, 200 μm Leupeptin, 360 mM NaCl pH 8.0 IEX SCRUB 10 mM Tris, 3 mMCaCl₂, 1.5M NaCl pH 8.0 FORMULATION 10 mM Tris, 60 mM Sucrose pH 8.03.0M AMMONIUM 10 mM Tris, 3.0M Ammonium Sulphate SULPHATE STOCK pH 8.0

TABLE 47 pH and Repeat testing Number Original conductivity of pH andcond. Date Date of of days buffer pH of retain Of retain Buffer Bufferprepared testing elapsed and cond. samples samples appearance HIC A 2May 2006 15 May 2006 13 days pH 7.98 pH 8.10 pH 7.89 Clear 127.6 137.5134.9 mS @17.8° C. mS@15.8° C. mS @ 22.0° C. HIC A2 2 May 2006 15 May2006 13 days pH 8.03 pH 8.05 pH 7.85 Clear 52.7 49.7 49.6 mS@18.5° C. mS@ 15.1° C. mS @ 22.0° C. HIC B 2 May 2006 15 May 2006 13 days pH 8.06 pH7.78 pH 7.81 Clear 0.699 0.879 0.842 mS@ 19.4° C. mS @ 16.7° C. mS@22.1° C. Diafiltration 2 May 2006 15 May 2006 13 days pH 8.05 pH 7.70N/A Clear buffer 0.668 0.793 mS@ 19.2° C. mS @ 18.2° C. IEX A 3 May 200615 May 2006 14 days pH 8.00 pH 7.69 N/A Clear 1.585 1.646 mS@ 20.3° C.mS @ 18.2° C. IEX B 3 May 2006 15 May 2006 14 days pH 8.00 pH 7.87 N/AClear 34.6 37.6 mS@ 19.4° C. mS @ 18.8° C. IEX Scrub 3 May 2006 15 May2006 14 days pH 8.00 pH 7.95 N/A Clear 105.2 122.9 mS@ 19.0° C. mS@18.3°C. Formulation 3 May 2006 15 May 2006 14 days pH 7.97 pH 7.89 N/A Clear0.735 0.987 mS@ 18.5° C. mS @19.1° C. 3.0M AS 3 May 2006 15 May 2006 14days pH 7.95 pH 7.96 N/A Clear Stock 251.0 255 mS@ 16.5° C. mS @19.9° C.3.0M AS 4 May 2006 15 May 2006 15 days pH 8.00 pH 7.97 N/A Clear Stock224.0 254 mS@ 14.6° C. mS @20.4° C.

Contaminant Identification by N-Terminal Sequencing

Three main impurities were detected for Process 3 by SDS-PAGE analysis.These appeared to be co eluted with the AUXI and AUXII collagenases andwere only resolved by fractionation of the peaks eluted from theQ-Sepharose column. The contaminants were assigned by their apparentmolecular mass on SDS-PAGE as 40 kDa, 55 kDa and 90 kDa contaminants.Fractions with elevated levels of a particular contaminant weresubmitted for N-terminal sequencing after excision of the band fromSDS-PAGE.

Sequence analysis was successful for both the 55 kDa and 40 kDacontaminant isolated from the 20 L demonstration run. The N-terminus ofthe 55 kDa contaminant band associated with AUXI (Lanes 1-5; FIG. 89)was shown to match a region of the Col G sequence for collagenase AUXIwhereas the 40 kDa contaminant band from AUXII (Lanes 6-10; FIG. 89) wasidentical to a region of the Col H sequence for collagenase AUXII. Aprevious attempt was made to sequence the 90 kDa band associated withboth the AUXI and AUXII products (FIG. 90). Sequencing of the 90 kDacontaminant associated with the AUXI product was successful in thatidentity was correlated with the N-terminus of the AUXI sequence. Incontrast, it was not possible to obtain a complete sequence for the 90kDa contaminant associated with AUXII, which suggested that the two 90kDa contaminants were different products.

The main contaminants associated with Process 3 appeared to be productrelated and were either identified as N-terminally cleaved products ofAUXI (55 kDa) and AUXII (40 kDa) or a C-terminally cleaved product ofAUXI (90 kDa). As these contaminants were different to those identifiedin Process 2, the QC assays utilized for the specification of theintermediates and drug substance did not resolve the new contaminants asthe assay development had originated around Process 2. In particular,the standard purity assay (RP-HPLC) could not be used to detect levelsof the 40 kDa and 55 kDa contaminants.

Densitometry Analysis 20 L Demonstration Run

The 40 kDa, 55 kDa and 90 kDa contaminants associated with Process 3were identified and resolved by SDS-PAGE. These contaminants wereclearly detected in fractions eluted from the Q-Sepharose column andappeared to elute at the leading and trailing edges of the peak profile(see FIGS. 72-76). The decision for which fractions were included orexcluded for further purification was based on experience of therelative intensity of staining for contaminants and product on Colloidalblue stained gels. In order to make this a less subjective estimation,densitometry was utilized to determine specific pooling criteria forfractions following the Q-Sepharose step. Densitometry was used inpreference to the current QC assay for purity (RP-HPLC) since this assaycould not resolve the new contaminants associated with Process 3.

Densitometry Data from Post Q Fractions from the 20 L Demonstration Run

The densitometry values from 2 separate analyses of the post-IEXfractions were averaged and are shown in table 48. Fractions 1-12 andthe last 25% (tail) of peak 1 contain AUXII and the associatedcontaminating proteins of 40, 75 and 90 kDa. Fractions 13-27 and thelast 25% (tail) of peak 2 contain AUXI and the associated contaminantsof 55 and 90 kDa. The pools of the fractions selected, based on SDS-PAGEwithout densitometric analysis, are highlighted.

TABLE 48

Densitometry Summary Documents from Post Q Fractions from the 200 LDemonstration Run

Summary of Densitometry Analysis

Post IEX fractions from the 200 L engineering run have been analyzedmultiple times to establish a pooling criteria that can be documented inthe IEX BMR for the GMP campaign. This pooling criterion is based on theassumption that: (i) the quality of material generated from theengineering run is appropriate for the GMP material; and (ii) theapproximation of the densitometry method is acceptable. If the aim is togenerate material of higher quality in the GMP campaign, thespecification for pooling criteria will need to be revised.

Specification for Pooling from the IEX

In total, the samples from the 200 L engineering run have been analyzed6 times (2 operators and 3 repeats of each gel) and the average datapresented in table 49. The fractions that were pooled for theengineering run are highlighted in red.

From this analysis, the following pooling criteria can be established:

(i) Any fraction of purity greater than or equal to 88.5% can be pooled.(ii) Any fraction with a single impurity greater or equal to 10% cannotbe pooled.(iii) Fractions to be pooled must be from consecutive fractions.(iv) The calculated theoretical purity of the pool should be:Greater than or equal to 93% theoretical purity for AUXI;Greater than or equal to 96% theoretical purity for AUXII.

This last point was based on the estimates from the 200 L engineeringrun in which the total protein in available fractions was estimated(although one limitation was that not all fractions were present for UVanalysis for the AUXI). The data from this analysis is presented intable 50.

**NOTE: from these criteria, fraction 7 for AUXII peak would now beexcluded.

Assay Variation

From the data of the post IEX fractions from the 200 L engineering run,the following level of accuracy has been estimated:

(i) for the product (AUXI and AUXII) the % CV had been calculated as2.1% (AUXI) and 2.3% (AUXII). Therefore the purity specification of88.6% for pooling could be in the range 86.3-90.9%.(ii) for the impurities, the % CV is much greater and the range has beenestimated as 18.5%-33.7% depending on the impurity. Consequently, thepurity specification of excluding fractions with a single impurity of nogreater than 10% could be for fractions with an actual impurity range of6.63-13.37%. Therefore the value for the purity of the product (and notthe impurities) is the most reliable value for pooling specification.

Estimated Purity of Final Material by Densitometry

Densitometry analysis of the final material (DS and intermediates) forthe 200 L engineering run has also been determined by densitometry andis as follows:

AUXI=96.0% (3.1% of 90 kDa contaminant)

AUXII=98.7% (1.2% of 90 kDa contaminant)

DS=97.6% (2.1% of 90 kDa contaminant)

(Note: This is the range determined for a single SDS-PAGE analysed 3times by 3 different operators.)

Standardisation of the Method

Over the course of the repeat analysis, the densitometry method has beenstandardized to minimize error between operators and variation betweengels and will be documented in an SOP. Most notably:

-   -   (i) The standard loading of total protein in each lane of the        gel will be 1 μg.    -   (ii) A maximum of 16 fractions will be selected for analysis        from each of the product peaks (AUXI and AUXII). This will limit        the number of gels for densitometry analysis to 4.    -   (iii) The 16 fractions selected will start at the last fraction        to be collected for each peak and work forward consecutively.        This is to ensure accuracy in the figure calculated for the        average purity (since all fractions to be pooled are likely to        be included).

TABLE 49 Average relative quantities of product and impurities in thepost IEX fractions from the 200 L Engineering run, as determined bydensitometry analysis. The fractions pooled are highlighted in red.Fraction # Quantity AUXI loaded Relative quantity (%) (peak 2) (μg)Product 40 90 80 55 3 1 88.91 0 7.43 0 3.66 4 1 85.81 0 7.28 0 6.91 5 184.57 0 6.78 0 8.65 6 1 80.41 0 6.96 0 12.63 8 1 80.00 0 5.46 0 14.54 91 80.61 0 5.53 0 13.86 10 1 88.53 0 4.20 0 7.27 11 1 90.43 0 4.19 0 5.3913 1 94.68 0 4.35 0 0.97 14 1 94.10 0 4.94 0 0.96 15 1 93.93 0 5.18 00.89 16 1 93.57 0 5.83 0 0.60 18 1 92.03 0 7.97 0 0 19 1 91.40 0 8.60 00 20 1 90.30 0 9.70 0 0 21 1 90.14 0 9.86 0 0 Quantity AUXII loadedRelative quantity (%) (peak 1) (mg) Product 40 90 80 55 3 1 68.48 21.559.97 0 0 4 1 55.51 36.80 7.69 0 0 5 1 55.90 36.73 4.96 2.41 0 6 1 67.5925.09 4.53 2.79 0 7 1 80.70 12.33 4.41 2.55 0 8 1 87.61 5.18 4.58 2.62 09 1 100 0 0 0 0 10 1 100 0 0 0 0 11 1 100 0 0 0 0 12 1 100 0 0 0 0 13 195.59 0 2.92 1.49 0 14 1 93.56 0 4.12 2.31 0 15 1 91.90 0 5.05 3.05 0 161 91.45 0 4.88 3.67 0 17 1 89.95 0 5.57 4.48 0 18 1 87.85 0 7.03 5.11 0

TABLE 50 Theoretical relative amounts of product and impurities in thepost IEX pools from the 200 L Engineering run, as determined bydensitometry analysis. The fractions pooled are highlighted in red. Thetheoretical average product purity is calculated as 92.3% and 95.8% forthe AUXI and AUXII intermediates respectively. Product Total protein(AUXI) Quantity of all quantity (mg) quantity (mg) Impurities (mg) AUXI954.09 844.67 109.42 1290.61 1167.07 123.55 1524.29 1443.16 81.131507.35 1418.46 88.89 1339.47 1258.18 81.29 1256.06 1175.32 80.741063.30 978.57 84.73 972.75 889.05 83.70 774.62 699.46 75.15 588.06530.06 58.00 Total 11270.60 10404.00 866.60 % 92.31 7.69 Product Totalprotein (AUXII) Quantity of all quantity (mg) quantity (mg) Impurities(mg) AUXII 200.20 161.56 38.64 323.14 283.11 40.02 533.02 533.02 0.00882.09 882.09 0.00 1226.58 1226.58 0.00 1508.94 1508.94 0.00 1206.731153.47 53.27 943.48 882.75 60.73 684.41 628.99 55.43 537.35 491.4045.95 312.80 281.35 31.45 348.53 306.19 42.34 Total 8707.28 8339.45367.82 % 95.78 4.22

GMP Pooling Criteria for Post Q-Sepharose Fractions Detail to beSpecified in the Ion Exchange BMR

A. The following pooling criteria is to be specified for fractions fromboth the AUXI and AUXII peaks which have been analyzed by densitometry:

-   -   (i) A maximum of 16 fractions will be selected for analysis from        each of the product peaks (AUXI and AUXII). This will limit the        number of gels for densitometry analysis to 4.    -   (ii) Any fraction of purity greater than or equal to 90.00%        (reported to 2 decimal places) can be pooled.    -   (iii) Any fraction with a single impurity greater than or equal        to 9.00% (to 2 dp) cannot be pooled.    -   (iv) Fractions to be pooled must be from consecutive fractions.        B. The following pooling criteria is to be specified for        fractions from the AUXII peak which have been analyzed by        SEC-HPLC.

(i) The maximum number of samples to be submitted for SEC-HPLC is 10 andmust be from the last fraction collected for this peak and consecutivefractions forward.

(ii) Any fraction with greater than or equal to 2.00% (to 2 dp)aggregate cannot be pooled.

Details to be Recorded for Information Only

A. The estimated theoretical purity of the pool should be calculated forinformation only and is expected to be:

Greater than or equal to 93.00% theoretical purity for AUXI;

Greater than or equal to 97.00% theoretical purity for AUXII.

B. The minimum quantity of protein in each pool should be noted toestablish if criteria for excluding fractions with less than 0.5 g couldbe used in the future.C. Fractions for the AUXI peak will be submitted for RP-HPLC but will beanalyzed retrospectively and for information only. These data will NOTbe considered as part of the pooling criteria.

Expected Impact on Pooling

The following has been calculated from the average data set presented intable 51 to reflect the effect on yield and fraction selection followingthe new pooling criteria:

TABLE 51 AUXI AUXII Fractions pooled from the 200 L engineering run#10-21 #7-18 Total protein determined for the post IEX pools from 13.18g 8.37 g the 200 L engineering run Fractions which would be pooledfollowing GMP #11-21 #9-16 criteria Estimated reduction in total proteindue to fractions  0.95 g 1.18 g excluded by the GMP criteria

TABLE 52 Average relative quantities of product and impurities in thepost IEX fractions from the 200 L Engineering run, as determined bydensitometry analysis. Fraction # Quantity AUXI loaded Relative quantity(%) (peak 2) (μg) Product 40 90 80 55 3 1 88.91 0 7.43 0 3.66 4 1 85.810 7.28 0 6.91 5 1 84.57 0 6.78 0 8.65 6 1 80.41 0 6.96 0 12.63 8 1 80.000 5.46 0 14.54 9 1 80.61 0 5.53 0 13.86 10 1 88.53 0 4.20 0 7.27 11 190.43 0 4.19 0 5.39 13 1 94.68 0 4.35 6 0.97 14 1 94.10 0 4.94 0 0.96 151 93.93 0 5.18 0 0.89 16 1 93.57 0 5.83 0 0.60 18 1 92.03 0 7.97 0 0 191 91.40 0 8.60 0 0 20 1 90.30 0 9.70 0 0 21 1 90.14 0 9.86 0 0 QuantityAUXII loaded Relative quantity (%) (peak 1) (mg) Product 40 90 80 55 3 168.48 21.55 9.97 0 0 4 1 55.51 36.80 7.69 0 0 5 1 55.90 36.73 4.96 2.410 6 1 67.59 25.09 4.53 2.79 0 7 1 80.70 12.33 4.41 2.55 0 8 1 87.61 5.184.58 2.62 0 9 1 100 0 0 0 0 10 1 100 0 0 0 0 11 1 100 0 0 0 0 12 1 100 00 0 0 13 1 95.59 0 2.92 1.49 0 14 1 93.56 0 4.12 2.31 0 15 1 91.90 05.05 3.05 0 16 1 91.45 0 4.88 3.67 0 17 1 89.95 0 5.57 4.48 0 18 1 87.850 7.03 5.11 0

TABLE 53 Theoretical relative amounts of product and impurities in thepost IEX pools from the 200 L Engineering run, as determined bydensitometry analysis. The fractions pooled are highlighted in red. Thetheoretical average product purity is calculated as 92.3% and 95.8% forthe AUXI and AUXII intermediates respectively. Product Total protein(AUXI) Quantity of all AUXI quantity (mg) quantity (mg) Impurities (mg)#10 954.09 844.67 109.42 #11 1290.61 1167.07 123.55 #13 1524.29 1443.1681.13 #14 1507.35 1418.46 88.89 #15 1339.47 1258.18 81.29 #16 1256.061175.32 80.74 #18 1063.30 978.57 84.73 #19 972.75 889.05 83.70 #20774.62 699.46 75.15 #21 588.06 530.06 1858.00 Total 11270.60 10404.00866.60 % 92.31 7.69 Product Total protein (AUXII) Quantity of all AUXIIquantity (mg) quantity (mg) Impurities (mg)  #7 200.20 161.56 38.64  #8323.14 283.11 40.02  #9 533.02 533.02 0.00 #10 882.09 882.09 0.00 #111226.58 1226.58 0.00 #12 1508.94 1508.94 0.00 #13 1206.73 1153.47 53.27#14 943.48 882.75 60.73 #15 684.41 628.99 55.43 #16 537.35 491.40 45.95#17 312.80 281.35 31.45 #18 348.53 306.19 42.34 Total 8707.28 8339.45367.82 % 95.78 4.22

A comparison of 2 data sets (i.e. the same in-process samples run ondifferent gels by different operators) allowed the followingretrospective pooling criteria to be noted for the average data setalthough one additional fraction (fraction 27 from AUXI) would beincluded from those actually pooled in the 20 L run:

-   -   AUXI        Pool all fractions with a purity of ≧87% but which do not have a        single impurity of ≧10%.    -   AUXII        Pool all fractions with a purity of ≧94% but which do not have a        single impurity of ≧4%.

QC data from the analysis of the final material from the 20 Ldemonstration run showed that the AUXII intermediate was 99.4% pure, theAUXI intermediate was 99.1% pure and the drug substance was 99.9%collagenase by RP-HPLC. Therefore, the criteria specified for thepooling process would be expected to result in material that passes therelease specifications for the final material.

200 L Demonstration Run

The criteria established for the 20 L demonstration run previouslymentioned was different to that implemented for the 200 L engineeringrun. In this case, pooling was specified for both the AUXI and AUXIIproducts as fractions with a purity of ≧86.5% but which did not have asingle impurity contaminant of ≧10%. AUXII samples with an impuritylevel ≧2% detected by SEC-HPLC for were also excluded. The resultingAUXI/AUXII intermediates and drug substance were also analyzed bydensitometry, using a standardized method, and shown to have thefollowing estimated purity based on analysis of a single gel 3 times (3different operators): AUXI=96.0% (3.1% of 90 kDa contaminant);AUXII=98.7% (1.2% of 90 kDa contaminant); DS=97.6% (2.1% of 90 kDacontaminant).

In addition, the QC determined purity of the intermediates and drugsubstance was show to pass specification by the RP-HPLC assay(AUXI=98.2%; AUXII=98.1%; drug substance=99.4%). Consequently, thepooling criteria followed for the 200 L engineering run was successfulin delivering product of suitable purity based on the current availableanalytical methods.

Materials and Methods

MUSTANG Q Chromatography (20 L scale run)

Equipment: MUSTANG Q Chromatography Capsule, 60 mL (CL3MSTGQP1, Pall)Conductivity and pH Meter 4330 (Jenway) Chemicals:

Sodium chloride (USP grade, Merck)Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)Tris (hydroxymethyl) methylamine (USP grade, Merck)Ammonium sulphate (Extra Pure, Merck)

Hyclone Water for Injection-Quality Water (WFI-QW)

A 60 mL bed volume MUSTANG Q chromatography capsule was sanitized with1M NaOH at a flow rate of 30 mL/min for 30 minutes. The capsule was thenpreconditioned for the same time and flow-rate using 1M NaCl. Thecapsule was equilibrated with 2 L of MUSTANG Q Equilibration buffer (10mM Tris, 1M ammonium sulphate, pH 8), at a flow rate of 60 mL/min. Theoutlet flow was checked to ensure the pH was <8. Supernatant (22 L) from200 L fermentation PP3 (which had been 0.2 μm filtered) was loaded ontothe capsule at a flow rate of 540 mL/min (approximately 40 min.duration). The maximum recommended operating flow rate for the capsulewas 600 mL/min. The filtered material was stored in 2×10 L Stedim bagsat 2-8° C. overnight.

Hydrophobic Interaction Chromatography (20 L Scale Run) Equipment:

AKTA Pilot installed with Unicorn V 5.01 software (GE Healthcare)Vantage 5130 column (cross sectional area 125 cm₂, Millipore)

Conductivity and pH Meter 4330 (Jenway)

Sartopore 2 0.8+0.45 μm filter capsule (Sartorius)

Medical Refrigeration Unit MP 150 (Electrolux) Chemicals: PhenylSepharose 6 FF low sub (GE Healthcare)

Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)Sodium chloride (USP grade, Merck)Tris(hydroxymethyl)methylamine (USP grade, Merck)Ammonium sulphate (Extra Pure, Merck)

Leupeptin (MP Biomedicals, Inc) Hyclone Water For Injection-QualityWater (WFI-QW) HIC Column Packing

2400 mL of Phenyl Sepharose 6 FF Low Sub (Lot#312089) slurry was settledfor 3 hours and the ethanol removed and replaced with 1800 mL WFI. Themedia was reslurried (50%), settled and washed once with WFI and twicewith 1800 mL 200 mM NaCl, with settling overnight between washes. Themedia was reslurried with 1800 mL 200 mM NaCl, poured into the columnand allowed to settle for 1 h. The adaptor was brought down to ˜1 cmabove the resin bed (removing all air bubbles) and the media packed in200 mM NaCl at a flow rate of 400 mL/min (192 cm/hr) for 10 mins. Thispacking flow rate was utilized as equivalent to the maximum operatingflow rate for the K-prime system available in GMP. The adaptor wasbrought down to the top of the bed and the column packed at 192 cm/hrfor 10 mins before screwing the adaptor into the top of the resin andpacking at 192 cm/hr for a further 10 mins, during which no compressionof the resin was observed. The pack test was carried out using the AKTAPilot method: HIC 1500 mL Pack Test. For this, the column wasequilibrated with 1 column volume (CV) of 200 mM NaCl in WFI and packtested with 15 mL (1% CV) of 1M NaCl in WFI at a flow rate of 313 mL/min(150 cm/hr). The column was flushed with 2CV WFI and stored with 2CV 10mM NaOH. The packed column had an asymmetry of 1.2, a plate count of2659 plates/meter, a CV of 1525 mL and bed height of 12.2 cm.

Column Sanitisation and Equilibration

The Phenyl Sepharose 6 FF (low sub) column was sanitized with 0.5M NaOHfor 60 minutes, washed with 2 column volumes (CV) WFI and equilibratedwith 5CV 10 mM Tris, pH 8 (HIC Buffer B) followed by 5CV 10 mM Tris,1.0M ammonium sulphate, pH 8 (HIC Buffer A).

Preparation of the HIC Load

13.48 kg (11.05 L) of 3.0M ammonium sulphate, 10 mM Tris, pH 8 was addedto 22.1 kg fermentation filtrate after the MUSTANG Q filter treatment(section 3.1). The filtrate was mixed for 5 minutes before filteringthrough a 0.05 m2 filter capsule (0.8+0.45 μm). The filtered material(denoted the HIC load material) was stored on ice (approximately 30minutes duration) until use.

HIC Column Run

The HIC run was performed at a constant linear flow rate of 150 cm/hourusing chilled buffers maintained at 2-8° C. 30 L feedstock (equivalentto 20 L post-MUSTANG Q filtrate) was loaded onto the 1525 mL PhenylSepharose 6 FF (low sub) column previously equilibrated with 2CV 10 mMTris, 1.0M ammonium sulphate, pH 8 (HIC Buffer A). Unbound material waswashed off the column with 10CV HIC Buffer A. The column was then washedwith 5CV 10 mM Tris, 0.3M ammonium sulphate, pH 8 (HIC Buffer A2) andbound proteins eluted with 10CV 10 mM Tris, pH 8 (HIC Buffer B). Thefirst 0.67 CV (1 L) of the elution buffer was discarded and a post-HICpool of 4CV was collected. Leupeptin was added (126.4 mL) to thepost-HIC pool (6191.3 g) to a final concentration of 200 μM from a stocksolution of 10 mM leupeptin, 10 mM Tris, pH 8. The mixed solution (6.3kg) was stored at 2-8° C. for 2 days before further processing bytangential flow filtration.

Tangential Flow Filtration Step 1 (TFF1 20 L Scale Run) Equipment:

ProFlux M12 TFF system (Millipore)Conductivity and pH meter 4330 (Jenways)Sartopore 2 0.8+0.45 μm filter capsule (Sartorius)Pellicon 2 “Mini” Filter 0.1 m₂ 30 kDa MWCO PES membranes (Millipore)

Medical Refrigeration Unit MP150 (Electrolux) Materials/Chemicals:

Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)Tris(hydroxymethyl)methylamine (USP grade, Merck)

Leupeptin (MP Biomedicals, Inc) Hyclone Water For Injection QualityWater (WFI-QW) System Step Up

The ProFlux M12 TFF system was set up according to the manufacturer'sinstructions with two Pellicon 2 “Mini” Filter 30 kDa MWCO PESmembranes, sanitised with 0.5M NaOH for 60 minutes and stored in 0.1MNaOH until use. The system was drained and flushed with 14 L WFI and thenormal water permeability (NWP) measured as 23 L/m2/hr/psi at 25° C. ata trans-membrane pressure (TMP) of 15 psig (inlet pressure of 20 psigand outlet pressure of 10 psig). The system was flushed with 0.5 L 10 mMTris, pH 8 (diafiltration buffer) and equilibrated with 1 L of the samebuffer for 10 minutes. The conductivity and pH of the permeate wasdetermined and checked against that of the diafiltration buffer toensure the membranes were equilibrated prior to use.

Concentration and Diafiltration

The concentration and diafiltration steps were performed with chilleddialfiltration buffer (10 mM Tris, pH 8) containing 200 μM leupeptin.The TFF system was flushed with 1 L chilled buffer just before use. 2 Lof the post-HIC material (6.3 L total volume) was pumped into the TFFsystem reservoir and recirculated for 10 minutes without back-pressureto condition the membrane. The level sensor on the reservoir was set to1.2 L and the post-HIC material concentrated at a TMP of 15 psig (inletpressure of 20 psig and outlet pressure of 10 psig) until all thematerial had entered the system. The permeate was collected and storedat 2-8° C. for analysis. The inlet tubing was connected to thediafiltration buffer and diafiltration of the material was performed ata TMP of 15 psig (inlet pressure of 20 psig and outlet pressure of 10psig) for approximately 8.5 turnover volumes (TOV), maintaining thevolume of material in the reservoir at 1.2 L. The conductivity and pH ofthe permeate was determined after 5, 7 and 8.5 TOV and checked againstthat of the diafiltration buffer. The retentate was drained from thesystem and stored at 2-8° C. 250 mL diafiltration buffer was pumped intothe reservoir, recirculated around the system for 10 minutes withoutbackpressure to rinse the system, drained, the rinse repeated and bothrinses were stored separately at 2-8° C. The protein concentration ofthe retentate and rinses were determined (by UV) and the first rinse(204.8 g weight) added to the retentate (1231.4 g weight). This postTFF1 material (1.4 kg) was then filtered through a Sartopore 2 0.8+0.45μm filter capsule and stored at 2-8° C. overnight until furtherprocessing by Q Sepharose ion exchange chromatography.

Ion Exchange Chromatography (20 L Scale Run) Equipment:

ÄKTA Pilot installed with Unicorn 5.01 software (GE Healthcare)

Conductivity and pH Meter 4330 (Jenway)

Vantage S90 Column (cross sectional area 62 cm₂, Millipore)

Medical Refrigeration Unit MP 150 (Electrolux) Chemicals:

Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)Sodium chloride (USP grade, Merck)Tris(hydroxymethyl)methylamine (USP grade, Merck)Calcium chloride 2-hydrate (USP grade, Merck)

Leupeptin (MP Biomedicals, Inc.) Q Sepharose HP (GE Healthcare) HycloneWater For Injection-Quality Water (WFI-QW) Column Packing andPreparation

A Vantage S90 column was packed using an AKTA Pilot chromatographysystem with Q Sepharose HP media in WFI to give a packed column with a10 cm bed height, therefore a column volume (CV) of 620 mL. The packingwas performed in accordance to the manufacturers instruction but withthe pressure limit of the Vantage column imposed (0.3 MPa) which equatedto a packing flow rate of 210 cm/hr and pressure limit of 0.28 MPa.After packing, the column was equilibrated with 2CV of 0.2M NaCl andpack tested with 1% CV (6.2 mL) 1M NaCl at a flow rate of 100 cm/hr (103mL/min). The packed column had an asymmetry of 1.6 and a plate count of12605 plates/meter, which was within specification for the media(asymmetry between 0.8 and 1.8, with a plate count >10,000). The columnwas stored in 10 mM NaOH until required.

Prior to use, the Q Sepharose column was washed with 1.5 column volumes(CV) of WFI to remove the storage buffer, sanitised with 0.5M NaOH for60 mins at 40 cm/hr before flushing again with 1.5CV WFI. The column wasthen charged and equilibrated in accordance to the manufacturersinstructions with 2CV 10 mM Tris, 3 mM calcium chloride, pH 8 followedby 2CV 10 mM Tris, 3 mM CaCl₂, 360 mM NaCl, pH 8 and finally 5CV 10 mMTris, 3 mM CaCl₂, pH 8.

Column Run

Immediately prior to the sample being loaded onto the column, the columnwas reequilibrated with chilled 10 mM Tris, 3 mM CaCl₂, 200 μM leupeptinpH 8 (IEX Buffer A). 1216 mL of chilled post TFF 1 material at aconcentration of 2.55 mg/mL (determined by UV) was loaded onto thecolumn at a flow rate of 100 cm/hr (103 mL/min). This equated to acolumn load of 5 mg total protein per mL of media. Following loading ofthe product, the column was washed with 3 column volumes (CV) of IEXBuffer A and the protein eluted with 10 mM Tris, 3 mM CaCl₂, 360 mMNaCl, 200 μM leupeptin, pH 8 (IEX Buffer B) with a gradient of 0-40%elution buffer (A to B), over 20CV at a flow rate of 70.2 ml/min (68cm/hr). Elution was monitored at 280 nm and 260 nm and 100 mL fractionscollected across the two product peaks containing AUX II and AUX I.Fraction collection was started from the breakthrough of the peak andcontinued until 25% of the peak height on the trailing edge. A total of12 fractions were collected across the AUX II peak and 15 fractionsacross the AUX I peak. The Q Sepharose HP chromatography was carried outat a standard laboratory temperature of 18-23° C., although the buffersused were pre-chilled. Fractions were stored at 2-8° C. until a resultwas obtained from the SDSPAGE analysis. Fractions 6 to 12 (peak 1) werepooled as AUX II collagenase with the volume determined as 683 g (aftersampling) and the concentration by UV analysis measured as 1.17 mg/mL.Fractions 19 to 26 (peak 2) were pooled as AUX I collagenase with thevolume determined as 796 g (after sampling) and the concentration by UVmeasured as 1.08 mg/mL.

Tangential Flow Filtration Step 2 (TFF2 20 L Scale Run) Equipment:

ProFlux M12 TFF system (Millipore)Conductivity and pH meter 4330 (Jenways)Pellicon 2 “Mini” Filter 0.1 m₂ 30 kDa MWCO PES membrane (Millipore)90 mm Filter Unit (1 L) 0.2 μm PES membrane (Nalgene)

Medical Refrigeration Unit MP150 (Electrolux) Materials/Chemicals:

Sodium hydroxide solution (volumetric 4M) (AnalaR, BDH)Tris(hydroxymethyl)methylamine (USP grade, Merck)Sucrose (BP grade, Merck)

Leupeptin (MP Biomedicals, Inc.) Hyclone Water For Injection-QualityWater (WFI-QW) Frensius Kabi Water For Injection (WFI) System Set Up

The ProFlux M12 TFF system was set up according to the manufacturer'sinstructions with one Pellicon 2 “Mini” Filter 30 kDa MWCO PES membrane,sanitized with 0.5M NaOH for 60 minutes and stored in 0.1M NaOH untiluse. The system was drained and flushed with 14 L WFI and the normalwater permeability (NWP) measured as 19.5 L/m₂/hr/psi for the membraneused for AUXI and as 14.5 L/m₂/hr/psi at 25° C. for the membrane usedfor AUXII at 25° C. and at a trans-membrane pressure (TMP) of 15 psig(inlet pressure of 20 psig and outlet pressure of 10 psig). The systemwas flushed with 0.5 L 10 mM Tris, 60 mM sucrose, pH8 (formulationbuffer), and equilibrated with 1 L of the same buffer for 10 minutes.The conductivity and pH of the permeate was determined and checkedagainst that of the formulation buffer.

Concentration and Formulation

The concentration and diafiltration steps were performed separately oneach of the post IEX pools of AUXI and AUXII. All steps were performedusing chilled formulation buffer (10 mM Tris, 60 mM sucrose, pH 8)maintained at 2-8° C. The TFF system was flushed with 1 L chilled bufferjust before use. The post-IEX pool (683 g weight of AUXII and 796 gweight of AUXI) was pumped into the TFF system reservoir andrecirculated at 10% pump speed for 10 minutes without backpressure tocondition the membrane. The level sensor on the reservoir was set toapproximately 400 mL and the AUXI or AUXII pool concentrated at a TMP of15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig) untilthe volume in the reservoir had been reduced to approximately 360-390 mL(this assumed a system hold up volume of 100 mL). The target volumereduction was based on achieving a theoretical concentration of 1.75mg/mL for the product assuming no loss in protein during theconcentration operation. The permeate was collected and stored at 2-8°C. for analysis. For the diafiltration operation the inlet tubing wasconnected to the formulation buffer and diafiltration performed at a TMPof 15 psig (inlet pressure of 20 psig and outlet pressure of 10 psig).Approximately 12 turnover volumes (TOV) were performed for AUXII and 8.5TOV's for AUXI, maintaining the volume of material in the reservoir at˜400 mL. The conductivity and pH of the permeate was determined after 12TOV for AUXII and after 6, 7, and 8.5 TOVs for AUXI and checked againstthat of the formulation buffer. The retentate was drained from thesystem and stored at 2-8° C. 250 mL formulation buffer was used to washresidual product from the membranes by re-circulated around the systemfor 10 minutes (without backpressure). After draining the rinsesolution, a second wash was performed and both rinse 1 and rinse 2 werestored at 2-8° C. After UV protein content determination of theretentate and rinses, the first rinse was added to the retentate, mixedand a UV protein concentration of the mix determined. For AUXII, 122 gof the second rinse was also added to the retentate plus rinse 1 to givea theoretical AUXII concentration of 1.1 mg/mL. For AUXI, 94 g of thesecond rinse was added to the material to give a theoretical AUXIconcentration of 1.1 mg/mL. Both the AUXI and AUXII material werefiltered through a 1 L Nalgene 0.2 μm filter unit in a Class II hood andthe post filtered protein concentration determined. The AUXI and AUXIIintermediates were stored at 2-8° C.

Protein Concentration Determination Absorbance Equipment: DU800Spectrophotometer (Beckman)

In process samples were analyzed by UV spectrophotometry by performing aUV scan of samples between 220 and 330 nm. The appropriate buffer wasused as a blank and a scan of the buffer blank performed before scanningthe samples. If necessary, samples were diluted with the same buffer toensure the A₂₈₀<1.0 AU. Protein concentrations (mg/mL) were determinedaccording to the Beer-Lambert law, c=A/b·ε, where A is the absorbance(A₂₈₀-A₃₃₀), b is the pathlength (1.0 cm) and c is the extinctioncoefficient of the protein. Extinction coefficients of 1.48 mg-1 cm-1 mLfor AUXI, 1.576 mg-1 cm-1 mL for AUXII and 1.428 mg-1 cm-1 mL for anAUXI/AUXII mix were used.

Bradford Assay Materials:

Lyophilised BSA (hydrated to 1.4 mg/mL)

Chemicals: Protein Assay Dye Reagent Concentrate (500-0006, Bio-Rad)

A BSA standard curve was prepared by diluting the BSA with water, toknown concentrations. The Bio-Rad protein assay dye reagent was preparedby diluting one part concentrate with four parts water. Test sampleswere prepared by diluting with water. 50 μL of test sample either neator diluted was added to a cuvette and 2.5 mL diluted regent added.Samples were prepared in duplicate. The samples were incubated for 10minutes before reading the OD. The standard curve of OD_(595 nm) vs.protein concentration was obtained by measuring the OD_(595 nm) of BSAsolutions of known concentration. The test samples were then assayed andthe protein concentration determined from the standard protein assaycurve. Samples from the post MUSTANG Q step were always analyzed withoutdilution in order to standardize the contribution from the pigment. Inthis case, 50 μL of the undiluted post MUSTANG Q material was utilizedin the assay.

SDS-PAGE Analysis Equipment: Xcell SureLock Mini-Cell ElectrophoresisSystem (Invitrogen) Electrophoresis Power Supply EPS 601, (AmershamPharmacia Biotech)

Rocky shaker platform, (Scientific Laboratory Supplies)

Chemicals: SDS-PAGE Standards High Molecular Weight (161-0303, Bio Rad)Mark12 Unstained Standard (LC5677, Invitrogen)

Novex 8% Tris-Glycine gels, 1.5 mm, 10 well (EC6018BOX, Invitrogen)NuPAGE Novex 4-12% Bis-Tris gels, 1.0 mm, 12 well (NP0322BOX,Invitrogen)

Novex Tris-Glycine SDS Running Buffer (10×) (LC2675, Invitrogen) NuPAGEMES SDS Running Buffer (20×) (NP0002, Invitrogen) Novex Tris-Glycine SDSSample Buffer (2×) (LC2676, Invitrogen) NuPAGE LDS Sample Buffer (4×)(NP0007, Invitrogen) NuPAGE Sample Reducing Agent (10×) (NP0009,Invitrogen) Colloidal Blue Staining kit (LC6025, Invitrogen)

Ethylenediaminetetra-acetic acid disodium salt AnalaR R (BDH)

Tris-Glycine Gels

Samples were prepared for reducing SDS-PAGE by adding 12 μl of sample to20 μl sample Buffer (2×), 4 μl reducing agent (10×) and 4 μl of 0.1MEDTA (to achieve final concentration of 10 mM). The high molecularweight (HMW) marker was prepared by adding 10 μl of concentrated stockto 80 μl reducing agent (10×), 310 μl WFI and 400 μl sample buffer (2×).The diluted HMW standard was then heated at 95° C. for 5 minutes beforealiquoting and storage at −20° C. for use in subsequent gels. Samples(20 μl load volume) containing collagenases were run directly (i.e. withno prior heat treatment) on 8% Tris-Glycine gels using Tris-Glycinerunning buffer at 130V for ˜2 hours. After electrophoresis, the gelswere stained with colloidal blue stain reagent as per the manufacturer'sinstructions.

Bis-Tris Gels

Samples were prepared for reducing SDS-PAGE by adding 16.5 μl of sampleto 7.5 μl sample buffer (4×), 3 μl reducing agent (10×) and 3 μl of 0.1MEDTA (to achieve final concentration of 10 mM). MARK 12 marker loadedneat (10 μl). Samples (15 μl load volume) containing collagenases wererun directly (i.e. with no prior heat treatment) on 4-12% Bis-Tris gelsusing either MES running buffer at 200V for ˜40 mins. Afterelectrophoresis, the gels were stained with either colloidal blue stainreagent as per the manufacturer's instructions or silver stained using astandard procedure (GE Healthcare).

Densitometry Analysis of Post-IEX Fractions Equipment: Xcell SureLockMini-Cell Electrophoresis System (Invitrogen) Electrophoresis PowerSupply EPS 601, (Amersham Pharmacia Biotech)

Rocky shaker platform, (Scientific Laboratory Supplies) Flatbed scanner(Hewlett Packard)

Materials/Chemicals:

NuPAGE Novex 4-12% Bis-Tris gels, 1.0 mm, 12 well (NP0322BOX,Invitrogen)

NuPAGE MES SDS Running Buffer (20×) (NP0002, Invitrogen) NuPAGE LDSSample Buffer (4×) (NP0007, Invitrogen) NuPAGE Sample Reducing Agent(10×) (NP0009, Invitrogen) Mark12 Unstained Standard (LC5677,Invitrogen) Colloidal Blue Staining kit (LC6025, Invitrogen)

Ethylenediaminetetra-acetic acid disodium salt (EDTA) (AnalaR, BDH)Purified water

Reducing SDS-PAGE

The post-IEX samples were run on 4-12% Bis-Tris gels using MES runningbuffer at 1 μg/lane loading. Samples were prepared by adding 20 μL ofdiluted post-IEX material to 8 μL Sample Buffer (4×), 3 μL ReducingAgent (10×) and 3.4 μL of 0.1M EDTA. 15 μL of each sample was loadedinto the well directly after mixing (i.e. with no heat treatment) andrun at 200V for 40 mins. After electrophoresis, the gels were stainedwith Colloidal Blue stain reagent according to the manufacturersinstructions but with a fixed staining duration to reduce stainingvariation (10 minute fix, 5 hours stain, 15-20 hours destain withpurified water).

Gel Scanning and Densitometry

Gels were placed between 2 sheets of acetate ensuring removal of all airbubbles, scanned on a flat-bed scanner at 600 dpi resolution and theimage cropped, resized and colour corrected with HP Image zone software.The image was converted to an 8-bit greyscale TIFF image with AlphaEaseFC software and the protein bands were analysed using QuantityOnegel documentation software (BioRad). After background substitution, theintensity peak areas of selected bands were converted to relativepercentage values of product (AUXI or AUXII) and impurity(s) in eachlane.

Buffer Stability Equipment: Peristaltic Pump (Watson Marlow)

125 ml PETG biotainers (Cellon)Watson Marlow Tubing for peristaltic pump

Conductivity and pH Meter 4330 (Jenway)

Sartopore 2 300 (0.45/0.2 μm) filter capsule (Sartorius)

Buffers for the 20 L demonstration run were filtered after preparationthrough a 0.45/0.2 μm filter capsule into 10 or 20 L Stedim bags forstorage at 2-8° C. prior to use. When the majority of the buffer hadbeen filtered, approximately 75 ml s of the remaining buffer wascollected into pre-labelled 125 ml PETG biotainers and stored at 2-8° C.The pH, conductivity, temperature and date of buffer preparation wererecorded. On completion of the 20 L demonstration run, the buffersamples were retrieved from cold storage and retested for pH,conductivity, and appearance. The temperature of the buffer at the timeof testing was also recorded.

Preparation of Samples for N-Terminal Sequencing Analysis Equipment:Electrophoresis Power Supply EPS 601, (Amersham Pharmacia Biotech) XcellSureLock Mini-Cell Electrophoresis System, (Invitrogen)

Rocky shaker platform, (Scientific Laboratory Supplies)

Chemicals:

Novex 8% Tris-Glycine Gel, 1.5 mm, 10 well, (Invitrogen)

High Molecular Weight Marker, (BioRad) NuPAGE Sample Reducing Agent(10×), (Invitrogen) Novex Tris-Glycine SDS Running Buffer (10×),(Invitrogen) Novex Tris-Glycine SDS Sample Buffer (2×), (Invitrogen)Colloidal Blue Staining Kit, (Invitrogen)

Ethylenediaminetetra-acetic acid disodium salt (EDTA) (AnalaR, BDH)

Methanol, AnalaR (BDH) Acetic Acid, AnalaR (BDH)

Water for injection (WFI)

Purified Water

Samples for N-terminal sequencing were prepared and separated on 8%Tris-Glycine gels as outlined previously. Samples identified as enrichedfor the 40 kDa contaminant (fraction 2 from the post IEX AUXII peak,CTL2006#0610H;) and 55 kDa contaminant (fraction 16 from the post IEXAUXI peak, CTL2006#0611H) were each loaded in 5 lanes of the gel toprovide enough material for sequencing (FIG. 89). Post IEX fractionsfrom a previous 20 L fermentation (20 L PP3), which were enriched forthe 90 kDa contaminants associated with both AUXI (fraction B7 R2,CTL2006#0581P) and AUXII (fraction D1, CTL2006#0582P) were also loadedin multiple lanes (FIG. 90). After electrophoresis, the gels werestained with colloidal blue stain reagent according to the manufacturersinstructions and the contaminant bands excised and submitted to AltaBioscience (Birmingham University, UK) for N-terminal sequencing. The 90kDa AUXI associated contaminant (CTL2006#0612H) from the 20 Ldemonstration run was also submitted for sequencing but no data wasobtained.

Summary of the Manufacturing of Process 3 Fermentation

The Phytone fed-batch fermentation process (Process 2) for production ofcollagenase from Clostridium histolyticum had been shown to be highlyvariable due to batch-to-batch variability in the Phytone peptone. Forthis reason Proteose Peptone #3 (PP3) was evaluated in 5 Lfermentations. The evaluation demonstrated that when one specific batchof PP3 was used at 50 g/L the fermentation process was robust andreproducible. However when other batches of PP3 were employed at 50 g/Llarge variations were seen in the growth profiles of the cultivations.The maximum biomass concentration the various batches of PP3 wouldsupport were assessed in a small scale evaluation. These batches weredeemed “good” or “poor” based on their ability to support high or lowbiomass concentrations of C. histolyticum respectively. When twofermentations were carried out at 5 L scale with “poor” and “good”batches of PP3 at 100 g/L both demonstrated highly similar growthprofiles and product yields. This experiment showed that increasing theconcentration of PP3 to 100 g/L alleviated the problem associated tobatch to batch variation in the peptone.

A scale up fermentation was carried out at 200 L. The fermentation usedthe optimized concentration of PP3 (100 g/L). The fermentation wassuccessful and replicated both the growth profile and productyield/quality observed at 5 L scale. The harvest process (clarificationby filtration) developed for Process 2 was evaluated during the 200 Lscale up fermentation. The cell culture was successfully clarified usingthe existing process with no blockage of the filtration train.

The quantification of collagenase concentration in crude fermentationsamples was improved using densitometry analysis of Coomassie stainedTris Glycine gels. A standard curve of mixed AUXI and AUXII was loadedwith dilutions of fermentation samples. The relationship betweencollagenase concentration and densitometry peak area was shown to belinear within the range of the sample dilutions. The concentrations ofcollagenase in the samples were then extrapolated using their peak areaand the standard curve. This method estimated the yield of collagenaseto be 280-350 mg/L from the 100 g/L PP3 process at 5 and 200 L scale.

The optimised PP3 fermentation process generated a higher biomassconcentration (OD600 7 units) and increased product yield (280-350 mg/Ltotal collagenase, by quantitative densitometry) when compared to thePhytone fed-batch process. The fermentation filtrate containedsignificantly less clostripain than the Phytone process. The ratio ofAUXI:AUXII was closer to 1 compared to that observed during evaluationof Process 2. In summary the PP3 process increased the product yield,purity (post-fermentation) and reproducibility of the fermentation.

Purification

Process 3 was developed in an accelerated time frame in order to improvethe processes previously developed at Cobra (Process 2) and run at 20 Lscale in GMP. Major improvements to the process were made in order tosimplify the purification procedure, facilitate robustness as well asmake the process more amenable to scale up to 200 L. These improvementswere also considered key to assisting process validation.

Process 3 was performed using material from a 200 L fermentation ofClostridium histolyticum in which a full 20 L of fermentation waspurified. Material was processed directly from the fermentation and nohold steps were implemented. Following filtration, product was passedthrough a MUSTANG Q filter since small-scale experiments demonstratedreduction of dsDNA (as detected by pico green analysis) using thisprocedure. Analysis of in-process samples from the 20 L demonstrationrun however, showed no reduction in dsDNA suggesting that the robustnessand application of this step required further investigation. Acomparison of the parameters used for the 20 L run-through and previoussmall-scale experiments demonstrated dsDNA removal when the capsule wasoversized by a factor of 1000 (based on the DNA binding capabilities of15-25 mg DNA/mL capsule described by the manufacturer). In comparison,the capsule used in the 20 L run-through was oversized by a factor ofapproximately 177-296. Material from the MUSTANG Q capsule was heldovernight at 2-8° C. An off-line stability study on sample materialtaken at this stage in the process indicated that maintaining a lowtemperature was key to the product stability at this point in theprocess since samples incubated at RT and 37° C. were susceptible todegradation as indicated by SDS-PAGE analysis.

Product from the MUSTANG Q capsule was prepared for hydrophobicinteraction chromatography (HIC) by the addition and mixing of anammonium sulphate solution (3M) to achieve a final concentration of 1M.This provided conditions suitable for collagenase binding to PhenylSepharose FF (low sub) media. A proportion of protein contaminants andpigment were then eluted from the HIC column using a step elution of0.3M ammonium sulphate followed by collagenase product elution with asolution containing no ammonium sulphate. Criteria for collection of theproduct peak were established as a fixed volume of 4 column volumes(although this was later extended to 5 column volumes for the 200 Lscale demonstration run). Leupeptin was then added immediately followingelution and the material held for a period of 2 days at 2-8° C.

The yield from this step was difficult to determine accurately due tothe complex nature of the feedstock. The process step yield wasestimated as (i) 38% based on Bradford assay of the load and UV of theeluted material or (ii) 47% based on collagenase content in the loadestimated by densitometry and UV of the eluted material. Alternatively,0.17 g of total protein was eluted from the HIC column for theequivalent of every 1 L of fermentation filtrate applied.

The post HIC pool was prepared for Q-Sepharose purification byconcentration (5-fold) and buffer exchange using tangential flowfiltration (TFF1) using 2×0.1 m2 30 kDa membranes. No loss was detectedover this step and the reported increase in protein recovered mayreflect the inaccuracy of UV at this point in the process. Inaccuracycould be attributed to pigment contamination or the use of theextinction coefficient for collagenases, which will be less accurate formaterial earlier in the purification when a complex of proteins arelikely to be present. The TFF step was completed by a product filtrationstep before holding the material at 2-8° C. over night.

As with Process 2, the Q-Sepharose column was a key purification step inProcess 3 and resulted in the separation of the AUXI and AUXIIcollagenases. The contaminants associated with process 3, however, weredifferent to those in process 2 and appeared to closely co-purify withthe AUXI and AUXII products. It was possible however, to remove thecontaminants from the products by fractionation of the product peakssince the contaminants appeared to elute at either the leading or tailedges of both peaks. The contaminants were denoted by their relativemolecular mass on reducing SDS-PAGE. Those associated with the AUXIIproduct (the first peak eluted from the Q-Sepharose column) wereidentified as (i) 40 kDa (associated with the leading edge of the peak)and (ii) 75 kDa and 90 kDa (associated with the trailing edge of thepeak). N-terminal amino acid sequencing indicated that the 40 kDa wasAUXII related since the sequence matched identity with a region of theCol H sequence. In comparison, no identity could be confirmed for the 90kDa contaminant due to issues of low signal. Contaminants associatedwith AUXI product (the second peak eluted form the Q-Sepharose column)were (i) 55 kDa (associated with the leading edge of the peak) and (ii)90 kDa (associated with the trailing edge of the peak). N-terminalsequencing showed both the 55 kDa and 90 kDa contaminants to beidentified as AUXI-related where the 55 kDa contaminant showed sequenceidentity with a mid region in the Col G sequence and the 90 kDa showedidentical N-terminal match to AUXI. Consequently, the major impuritiesidentified at this stage in the process were all product related andeither identified as internal cleavage products of AUXI (55 kDa) andAUXII (40 kDa) or a C-terminally cleaved product of AUXI (90 kDa).

Following the Q-Sepharose column, a key process step was in the decisionas to which fractions should go forward for further purification. Forthe 20 L demonstration run this criteria was based on the relativestaining intensities of contaminants to product when analyzed by 4-12%SDS-PAGE and stained with Colloidal Blue stain. The decision wassubjective and based on the collective experience of the processdevelopment group as well as requests from the client. In order toestablish defined criteria that described the pooling procedure,densitometry was performed on SDSPAGE. From this, the pooling wasdescribed as including fractions that were ≧87% pure (with no singleimpurity ≧10%) for AUXI and ≧94% pure (with no single impurity ≧4%) forAUXII. This resulted in a step yield based on UV estimation of 27.7% and25.8% for AUXI and AUXII respectively. Further refinement andstandardization of the densitometry method was achieved from dataacquired from the 200 L scale demonstration run which resulted indefinition of modified criteria for the subsequent GMP run.

Fractions containing AUXI or AUXII product from the Q-Sepharose columnwere formulated separately by TFF (denoted TFF2) using 1×0.1 m2 30 kDamembrane for each collagenase. The formulation buffer of 10 mM Tris, 60mM sucrose pH 8, had been established by KBI BioPharma Inc. Product wasfiltered following the TFF2 step and the overall step yields for TFF andfiltration estimated as 97.5% for AUXI and 92.2% for AUXII. At thisstage samples were referred to as intermediates and were retained at2-8° C. for QC analysis and prior to mixing of the drug substance. Aretrospective stability study indicated the intermediates were stableover a period of at least 5 days at 2-8° C. as determined by SDS-PAGE,UV, RP-HPLC and SEC-HPLC analysis. The only detected deterioration inintermediates was identified in the AUXII intermediate after a 12 dayhold in which aggregate levels increased from 0 to 0.62%.

The AUXI and AUXII intermediates were mixed in equal ratio (asdetermined by UV) to generate the drug substance before performing afinal product filtration. Only 400 mg of drug substance was prepared ofwhich 200 mg was shipped to KBI BioPharma Inc. along with 25 mg of eachintermediate. The overall process yield was estimated for the 20 Ldemonstration run in which all available material from the 20 L offermentation feedstock had been processed and assuming all material hadbeen mixed as drug substance. This gave a predicted yield of 1.6 g drugsubstance for the 20 L scale purification. This equated to a processrecovery of 17.8% based on then assumption that the initial estimate of9 g (using the Bradford assay) for the amount of total protein availableto load onto the HIC column was accurate. Alternatively, if the totalavailable protein was related to the collagenase content in the HIC load(as estimated by densitometry) the overall process yield was calculatedas 22%.

In addition to the process run-through, some preliminary studies werepreformed on sample and buffer retains taken from the process to assessstability. These data indicated that for the product, low temperaturewas a key factor in controlling degradation and samples taken early inthe purification (prior to the Q-Sepharose column) were more susceptibleto proteolysis. A product hold study showed however, that thecombination of leupeptin and temperature control (2-8° C.) wassuccessful in maintaining the product quality over the time coursesanticipated for the GMP process.

Tables 54 and 55 detailed the analytical specifications AUX-I and AUX-IIintermediates and also for Drug Substance for Process 3.

TABLE 54 Analytical Specifications for Process 3 AUX-I and AUX-IIIntermediates Specification Test AUX-I AUX-II Appearance Clear colorlessand free Clear colorless and free from particulate matter fromparticulate matter *Endotoxin ≦10 EU/mL ≦10 EU/mL Identity (and purity)by SDS- Major band between 98-188 kDa, Major band between 98-188 kDa,PAGE (Reduced conditions, and no minor and no minor Coomasie and silverstained) bands bands *Total Protein by Absorbance 0.8-1.2 mg/mL 0.8-1.2mg/mL Spectroscopy SRC assay (AUX-I) 12 000-21 000 fSRC Not applicableunits/mg GPA assay (AUX-II) Not applicable 370 000-680 000 fGPA units/mgAnalysis of Proteins using ≧98% main peak ≧98% main peak the Agilent1100 HPLC System (Aggregation by size exclusion chromatography)*Analysis of Proteins using the ≧97% by area ≧97% by area Agilent 1100HPLC System (Purity by reverse phase liquid chromatography) Analysis ofProteins using the ≦1% by area ≦1% by area Agilent 1100 HPLC System(Residual gelatinase by anion exchange chromatography) Analysis ofProteins using the ≦1% by area ≦1% by area Agilent 1100 HPLC System(Residual clostripain by reverse phase liquid chromatography) Identityby Peptide Mapping Conforms to reference Conforms to reference Bioburden≦100 CFU/mL ≦100 CFU/mL *Tests required for provisional release ofintermediates for further manufacturing

TABLE 55 Analytical Specifications for Process 3 Drug SubstanceSpecification Test AUX-I AUX-II Appearance Clear colorless andessentially free from particulate matter Potentiometric Measure of 7.5to 8.5 pH of Solution Endotoxin <10 EU/mL Identity (and purity) by MajorMajor collagenase band SDS-PAGE (Reduced collagenase between 97-200 kDa;conditions, Coomasie and band between MW markers; major silver stained)98-188 kDa bands comparable to MW markers reference standard *TotalProtein by 0.8-1.2 mg/mL Absorbance Spectroscopy *SRC assay (AUX-I) 13000-23 000 NA fSRC units/mg *GPA assay (AUX-II) NA 200 000-380 000 fGPAunits/mg Residual host cell protein Comparable to reference standard; noindividual impurity band exhibiting greater intensity than 1% BSAintensity marker Residual host cell DNA ≦10 pg/dose Analysis of Proteinsusing the ≧98% main peak; ≦2% aggregates Agilent 1100 HPLC System byarea (Aggregation by size exclusion chromatography) *Analysis ofProteins using 2 major peaks (AUX I & AUX II), the Agilent 1100 HPLCcombined ≧97% by area; Retention System (Identity and purity times ofAUX-I and AUX-II within by reverse phase liquid 5% of referencechromatography) Analysis of Proteins using ≦1% by area the Agilent 1100HPLC System (Residual clostripain by reverse phase liquid chromatographyAnalysis of Proteins using ≦1% by area the Agilent 1100 HPLC System(Residual gelatinase by anion exchange chromatography) Residualleupeptin by ≦1 ug/mg w/w reverse phase chromatography *Bioburden <1cfu/mL *Tests required for provisional release of Drug Substance forfurther manufacturing.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A drug product comprising isolated and purified collagenase I andcollagenase II having the sequence of Clostridium histolyticumcollagenase I and collagenase II, respectively, wherein the collagenaseI and collagenase II have a mass ratio of about 1 to 1 and the drugproduct is at least 95% by area pure as determined by reverse phase highperformance liquid chromatography.
 2. The drug product of claim 1,wherein the drug product contains less than about 2% by area aggregatedprotein as determined by reverse phase high performance liquidchromatography.
 3. The drug product of claim 1, wherein the drug productcontains less than about 1% by area of clostripain as determined byreverse phase high performance liquid chromatography.
 4. The drugproduct of claim 1, wherein the drug product contains less than about 1%by area of gelatinase as determined by anion exchange chromatography. 5.The drug product of claim 1, wherein the drug product contains less thanabout 1 ug/mg (w/w) of leupeptin.
 6. The drug product of claim 1,wherein the drug product has a bioburden less than 1 cfu/ml, and whereinthe drug product is sterile.
 7. The drug product of claim 6, wherein thedrug product contains less than 10 EU/ml of endotoxin.
 8. The drugproduct of claim 6, wherein the drug product contains less than 5 EU/mgof endotoxin.
 9. A drug product comprising isolated and purifiedcollagenase I and collagenase II having the sequence of Clostridiumhistolyticum collagenase I and collagenase II, respectively, wherein thecollagenase I and collagenase II have a mass ratio of about 1 to 1 andthe drug product is at least 95% by area pure as determined by reversephase high performance liquid chromatography, wherein the preparation ofthe drug product comprises the steps of: a) fermenting Clostridiumhistolyticum; b) harvesting a crude fermentation comprising collagenaseI and collagenase II; c) purifying collagenase I and collagenase II fromthe crude harvest via filtration and column chromatography comprisingthe steps of: i) filtering the crude harvest through an anion exchangefilter; ii) adding ammonium sulphate; iii) subjecting the harvestthrough a HIC column; iv) adding leupeptin to the filtrate; v) removingthe ammonium sulfate; vi) filtering the mixture of step (v); and vii)separating collagenase I and collagenase II using ion-exchange; d)combining the collagenase I and collagenase II purified from step (c) ata ratio of about 1 to
 1. 10. The drug product of claim 9, wherein thedrug product is at least 97% by area pure as determined by reverse phasehigh performance liquid chromatography.
 11. The drug product of claim 9,wherein preparation of the drug product further comprises the step ofconducting cell bank preparations in the presence of phytone peptone orvegetable peptone.
 12. The drug product of claim 9, wherein thefermentation step comprises the steps of: i. inoculating the medium in afirst stage with Clostridium histolyticum and agitating the mixture; ii.incubating the mixture from step (i) to obtain an aliquot; iii.inoculating the medium in a second stage with aliquots resulting fromstep (ii) and agitating the mixture; iv. incubating mixtures from step(iii) to obtain an aliquot; v. inoculating the medium in a third stagewith aliquots resulting from step (iv) and agitating; vi. incubatingmixtures from step (v) to obtain an aliquot; vii. inoculating the mediumin a fourth stage with an aliquot resulting from step (vi) andagitating; and viii. incubating mixtures from step (vii).
 13. The drugproduct of claim 9, wherein the drug product is stored at a temperatureof about −70° C. 14-18. (canceled)
 19. A pharmaceutical formulationcomprising a pharmaceutically acceptable excipient and a drug productcomprising isolated and purified collagenase I and collagenase II havingthe sequence of Clostridium histolyticum collagenase I and collagenaseII, respectively, wherein the collagenase I and collagenase II have amass ratio of about 1 to 1 and the drug product is at least 95% by areapure as determined by reverse phase high performance liquidchromatography.
 20. The pharmaceutical formulation of claim 19, whereinthe drug product is a sterile lyophilized powder and is stored at atemperature of about 5° C.
 21. The pharmaceutical formulation of claim19, wherein the formulation is a lyophilized injectable formulationformulated with Sucrose, Tris and with a pH level of about 8.0.
 22. Thepharmaceutical formulation of claim 21, wherein the formulation is alyophilized injectable formulation comprising about 0.9 mg of the saiddrug product, about 18.5 mg of sucrose and about 1.1 mg of Tris, andwherein the targeting vial fill volume is about 0.9 mL.
 23. Thepharmaceutical formulation of claim 21, wherein the formulation is alyophilized injectable formulation comprising about 0.58 mg of the saiddrug product, about 12.0 mg of sucrose and about 0.7 mg of Tris.
 24. Thepharmaceutical formulation of claim 19, wherein the preparation of thedrug product comprises the steps of: a) fermenting Clostridiumhistolyticum; b) harvesting a crude fermentation comprising collagenaseI and collagenase II; c) purifying collagenase I and collagenase II fromthe crude harvest via filtration and column chromatography comprisingthe steps of: i) filtering the crude harvest through an anion exchangefilter; ii) adding ammonium sulphate; iii) subjecting the harvestthrough a HIC column; iv) adding leupeptin to the filtrate; v) removingthe ammonium sulfate; vi) filtering the mixture of step (v); and vii)separating collagenase I and collagenase II using ion-exchange; d)combining the collagenase I and collagenase II purified from step (c) ata ratio of about 1 to
 1. 25. The pharmaceutical formulation of claim 24,wherein the formulation is a sterile lyophilized powder.
 26. Thepharmaceutical formulation of claim 25, wherein the formulation is alyophilized injectable formulation formulated with sucrose, Tris andwith a pH level of about 8.0.
 27. The pharmaceutical formulation ofclaim 26, wherein the formulation is a lyophilized injectableformulation comprising about 0.9 mg of the said drug product, about 18.5mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vialfill volume is about 0.9 mL.
 28. The pharmaceutical formulation of claim26, wherein the formulation is a lyophilized injectable compositioncomprising about 0.58 mg of the said drug product, about 12.0 mg ofsucrose and about 0.7 mg of Tris. 29-46. (canceled)
 47. Thepharmaceutical formulation of claim 19, wherein the drug product is atleast 97% by area pure as determined by reverse phase high performanceliquid chromatography.
 48. The pharmaceutical formulation of claim 47,wherein the formulation is a sterile lyophilized powder and is stored ata temperature of about 5° C.
 49. The pharmaceutical formulation of claim47, wherein the formulation is a lyophilized injectable formulationformulated with Sucrose, Tris and with a pH level of about 8.0.
 50. Thepharmaceutical formulation of claim 49, wherein the formulation is alyophilized injectable formulation comprising about 0.9 mg of the saiddrug product, about 18.5 mg of sucrose and about 1.1 mg of Tris, andwherein the targeting vial fill volume is about 0.9 mL.
 51. Thepharmaceutical formulation of claim 49, wherein the formulation is alyophilized injectable formulation comprising about 0.58 mg of the saiddrug product, about 12.0 mg of sucrose and about 0.7 mg of Tris.
 52. Thepharmaceutical formulation of claim 47, wherein the preparation of thedrug product comprises the steps of: a) fermenting Clostridiumhistolyticum; b) harvesting a crude fermentation comprising collagenaseI and collagenase II; c) purifying collagenase I and collagenase II fromthe crude harvest via filtration and column chromatography comprisingthe steps of: i) filtering the crude harvest through an anion exchangefilter; ii) adding ammonium sulphate; iii) subjecting the harvestthrough a HIC column; iv) adding leupeptin to the filtrate; v) removingthe ammonium sulfate; vi) filtering the mixture of step (v); and vii)separating collagenase I and collagenase II using ion-exchange; d)combining the collagenase I and collagenase II purified from step (c) ata ratio of about 1 to
 1. 53. The pharmaceutical formulation of claim 52,wherein the formulation is a sterile lyophilized powder.
 54. Thepharmaceutical formulation of claim 52, wherein the formulation is alyophilized injectable formulation formulated with sucrose, Tris andwith a pH level of about 8.0.
 55. The pharmaceutical formulation ofclaim 54, wherein the formulation is a lyophilized injectablecomposition comprising about 0.9 mg of the said drug product, about 18.5mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vialfill volume is about 0.9 mL.
 56. The pharmaceutical formulation of claim54, wherein the formulation is a lyophilized injectable compositioncomprising about 0.58 mg of the said drug product, about 12.0 mg ofsucrose and about 0.7 mg of Tris.